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The objective was to evaluate the analytical performance of a new point-of-need platform for rapid and accurate measurement of a host-protein score that differentiates between bacterial and viral infection. The system comprises a dedicated test cartridge (MeMed BV®) and an analyzer (MeMed Key®). In each run, three host proteins (TRAIL, IP-10 and CRP) are measured quantitatively and a combinational score (0-100) computed that indicates the likelihood of Bacterial versus Viral infection (BV score). Serum samples collected from patients with acute infection representing viral (0 ≤ score 35), equivocal (35 ≤ score ≤ 65), or bacterial (65 score ≤ 100) scores based on pre-defined score cutoffs were employed for the analytical evaluation studies as well as samples from healthy individuals. To assess reproducibility, triplicate runs were conducted at 3 different sites, on 2 analyzers per site over 5 non-consecutive days. Lower limit of quantitation (LLoQ) and analytical measurement range were established utilizing recombinant proteins. Sample stability was evaluated using patient samples representative of BV score range (0-100). MeMed Key® and MeMed BV® passed the acceptance criteria for each study. In the reproducibility study, TRAIL, IP-10 and CRP measurements ranged with coefficient of variation from 9.7 to 12.7%, 4.6 to 6.2% and 5.0 to 11.6%, respectively. LLoQ concentrations were established as 15 pg/mL, 100 pg/mL and 1 mg/L for TRAIL, IP-10 and CRP, respectively. In summary, the analytical performance reported here, along with diagnostic accuracy established in the Apollo clinical validation study (NCT04690569), supports that MeMed BV® run on MeMed Key® can serve as a tool to assist clinicians in differentiating between bacterial and viral infection.

The mnemonic SPICE ( Serratia , Pseudomonas , indole-positive Proteus , Citrobacter , and Enterobacter ) has served as a reminder to consider when a Gram-negative organism may carry a chromosomal copy of bla ampC , with the associated risk of developing resistance to first-, second-, and third-generation cephalosporins. However, in 2017, there was a well-founded proposal to rename Enterobacter aerogenes to Klebsiella aerogenes , based on whole-genome sequencing (WGS), and the SPICE mnemonic lost its relevance.

Background We have previously reported on a US based national surveillance study from 6 geographically dispersed medical centers for the susceptibility and epidemiology of C. difficile isolates. This current survey was conducted with isolates collected in 2020-21 with specific attention to the susceptibility to ridinilazole as well as nine comparators. A summary of the susceptibilities of 300 isolates of C. difficile tested against 10 antimicrobial agents Methods in 2020-21 C. difficile isolates or stools from patients with C. difficile toxin positive antibiotic associated diarrhea were collected in 6 US medical centers and referred to a central lab. After confirmation of 300 isolates antibiotic susceptibilities were determined by agar dilution method (CLSI M11-A9) against 10 agents listed in Table below. Ribotyping was performed by PCR capillary gel electrophoresis on all isolates. Results A summary of the susceptibilities of the 300 isolates against 10 antimicrobial agents are shown in the Table. The most common ribotype was 014-020 (14.4% compared to 11.8% in 2016), followed by 106 (10%, compared to 15% in 2016), 027 (10% compared to 13.1% in 2016), 002 (8% compared to 8.5% in 2016, 078-126 (4.3% compared to 1.3% in 2016). Ridinilazole was very potent against all isolates collected in the US with an MIC 90 of 0.25 mcg/ml, including against all the hypervirulent ribotypes as well as those isolates with antibiotic resistances to other antibiotics in the test panel. Conclusion Ridinilazole showed excellent in-vitro activity against C. difficile isolates in the US, independent of ribotype. There has been a striking change in ribotype distribution compared to 2016, accompanied by a reduction in C. difficile resistance to moxifloxacin and imipenem. Disclosures Cheleste M. Thorpe, MD, Actelion: Grant/Research Support|Deinove: Advisor/Consultant|General Mills: Grant/Research Support|Merck: Grant/Research Support|Pfizer: Grant/Research Support|Summit: Advisor/Consultant|Summit: Grant/Research Support Stuart Johnson, M.D., Ferring Pharmaceuticals: Membership on Ferring Publication Steering Committee|Ferring Pharmaceuticals: Employee|Summit Plc: Advisor/Consultant Dale N. Gerding, MD, Destiny Pharma plc.: Advisor/Consultant Karen C. Carroll, MD, Abbott Diagnostics: Advisor/Consultant|BD Diagnostics: Advisor/Consultant|BD Diagnostics: Grant/Research Support|BioRad, Inc: Grant/Research Support|Meridian Biosciences: Grant/Research Support|Roche Diagnostics: Advisor/Consultant|Scanogen, Inc: Advisor/Consultant|Scanogen, Inc: Grant/Research Support|Thermo Fisher, Inc: Advisor/Consultant Kevin W. Garey, PharmD, MS, Acurx: Grant/Research Support|cidara: Advisor/Consultant|cidara: Grant/Research Support|Paratek: Grant/Research Support|Seres Health: Grant/Research Support|Summit: Grant/Research Support Esther Duperchy, PhD, Summit Plc: Employee David R. Snydman, MD, Merck: Advisor/Consultant|Merck and Company: Grant/Research Support|Prolacta: Advisor/Consultant|Prolacta: Grant/Research Support|Seres: Advisor/Consultant|Seres Health: Grant/Research Support|Summit Plc: Grant/Research Support|Takeda: Advisor/Consultant|Takeda: Grant/Research Support|Visterra: Advisor/Consultant.

Background Hospitalizations due to soft tissue methicillin resistant S. aureus (MRSA) infections are decreasing nationally. Analyzing trends in pediatric community-onset (CO) S. aureus infections is needed to inform empiric antibiotic selection in the Emergency Department and outpatient settings. Methods We identified all cultures growing S. aureus between January 1, 2014 and December 31, 2020 in the Johns Hopkins Hospital microbiology laboratory that services an academic medical center, a mid-level teaching hospital, and a community hospital. Patients were included if they were ≤18 years of age or treated in a pediatric location. Cultures that likely represented colonization, such as nasal swabs, throat swabs, or sputum were excluded. Patients hospitalized within 2 calendar days of culture collection were defined as treated in-hospital. All others were defined as treated outpatient. CO infection was defined as a culture obtained within 3 days of hospitalization or from children not hospitalized. Only the first culture per patient per quarter was included. Cochran Armitage test was used to analyze trends in antibiotic susceptibilities. Results 3517 cultures obtained from 3078 patients were included. 3208 cultures (91%) were CO. Of CO cultures, 2517 (78.5%) were treated in an outpatient setting and 691 (21.5%) were treated in-hospital. There was a 74% decrease in the number of CO S. aureus cultures during the study period: 751 in 2014 to 191 in 2020. Comparing CO cultures from 2014 and 2020, methicillin susceptibility increased from 59.5% to 67% overall (p = 0.007), 59.4% to 63.8% in outpatient treated cultures (p = 0.07), and 60% to 76% among in-hospital treated cultures (p =0.02). Methicillin susceptibility from CO soft tissue sources remained about 60% during the study period with no significant change in trend. When soft tissue sources were excluded from CO cultures, methicillin susceptibility increased from 61.7% in 2014 to 84.1% in 2020 (p = 0.01). Within CO blood cultures, methicillin susceptibility increased from 86.7% to 92.3% (p = 0.4). Conclusion In a regional health system, CO pediatric MRSA incidence is decreasing. Although the majority of CO bloodstream S. aureus infections are methicillin susceptible, MRSA continues to cause up to 40% of S. aureus soft tissue infections. Disclosures Aaron Milstone, MD, Merck: Grant/Research Support Karen C. Carroll, MD, Abbott Diagnostics: Advisor/Consultant|BD Diagnostics: Advisor/Consultant|BD Diagnostics: Grant/Research Support|BioRad, Inc: Grant/Research Support|Meridian Biosciences: Grant/Research Support|Roche Diagnostics: Advisor/Consultant|Scanogen, Inc: Advisor/Consultant|Scanogen, Inc: Grant/Research Support|Thermo Fisher, Inc: Advisor/Consultant.

Background Our objective was to determine if the addition of ultraviolet-C (UV-C) light to daily and discharge patient room cleaning reduces healthcare-associated infection rates of vancomycin-resistant enterococci (VRE) and Clostridioides difficile in immunocompromised adults. Methods We performed a cluster randomized crossover control trial in 4 cancer and 1 solid organ transplant in-patient units at the Johns Hopkins Hospital, Baltimore, Maryland. For study year 1, each unit was randomized to intervention of UV-C light plus standard environmental cleaning or control of standard environmental cleaning, followed by a 5-week washout period. In study year 2, units switched assignments. The outcomes were healthcare-associated rates of VRE or C. difficile. Statistical inference used a two-stage approach recommended for cluster-randomized trials with Results In total, 302 new VRE infections were observed during 45787 at risk patient-days. The incidence in control and intervention groups was 6.68 and 6.52 per 1000 patient-days respectively; the unadjusted incidence rate ratio (IRR) was 0.98 (95% confidence interval [CI], .78 − 1.22; P = .54). There were 84 new C. difficile infections observed during 26118 at risk patient-days. The incidence in control and intervention periods was 2.64 and 3.78 per 1000 patient-days respectively; the unadjusted IRR was 1.43 (95% CI, .93 − 2.21; P = .98). Conclusions When used daily and at post discharge in addition to standard environmental cleaning, UV-C disinfection did not reduce VRE or C. difficile infection rates in cancer and solid organ transplant units.

BACKGROUND: Animal-assisted intervention (AAI) programs, used widely for patient benefit, have increasingly been used for healthcare workers (HCW) to reduce occupational stress. However, there are barriers to these programs which limit their utilization, for both patients and HCW, specifically infectious disease concerns. The aim of the research project is to identify barriers and facilitators to AAI program use for healthcare worker benefit, and determine knowledge, beliefs, and practices regarding infectious disease risk and control policies, in order to understand the contextual parameters of program implementation. METHODS: We collected perceptions of key stakeholders involved with hospital AAI programs (HCW and AAI workers) through semi-structured in-depth interviews. We used framework analysis to guide thematic coding, completed independently by three researchers. RESULTS: We interviewed 37 participants in this study. We divided our themes into two topic areas: program use for HCW and perceived infectious disease risk. Use for healthcare workers included perspectives on the benefits for HCW and program barriers and facilitators (specifically collaboration and leadership). Perceived risk included opinions on infection concerns with AAI, thoughts on control measures to reduce this risk, and responsibility for safety during these programs. CONCLUSIONS: While significant benefits were reported for HCW, they were limited by administrative barriers and hazard concerns. Facilitators to surmount these barriers are best implemented with collaboration across the hospital and appropriate leadership roles to direct safe program implementation. By addressing these barriers through targeted facilitators in the form of evidence-backed guidelines, AAI programs can be used to benefit both patients and HCW.

Next-generation sequencing (NGS) workflows applied to bronchoalveolar lavage (BAL) fluid specimens could enhance the detection of respiratory pathogens, although optimal approaches are not defined. This study evaluated the performance of the Respiratory Pathogen ID/AMR (RPIP) kit (Illumina, Inc.) with automated Explify bioinformatic analysis (IDbyDNA, Inc.), a targeted NGS workflow enriching specific pathogen sequences and antimicrobial resistance (AMR) markers, and a complementary untargeted metagenomic workflow with in-house bioinformatic analysis. Compared to a composite clinical standard consisting of provider-ordered microbiology testing, chart review, and orthogonal testing, both workflows demonstrated similar performances. The overall agreement for the RPIP targeted workflow was 65.6% (95% confidence interval, 59.2 to 71.5%), with a positive percent agreement (PPA) of 45.9% (36.8 to 55.2%) and a negative percent agreement (NPA) of 85.7% (78.1 to 91.5%). The overall accuracy for the metagenomic workflow was 67.1% (60.9 to 72.9%), with a PPA of 56.6% (47.3 to 65.5%) and an NPA of 77.2% (68.9 to 84.1%). The approaches revealed pathogens undetected by provider-ordered testing (Ureaplasma parvum, Tropheryma whipplei, severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2], rhinovirus, and cytomegalovirus [CMV]), although not all pathogens detected by provider-ordered testing were identified by the NGS workflows. The RPIP targeted workflow required more time and reagents for library preparation but streamlined bioinformatic analysis, whereas the metagenomic assay was less demanding technically but required complex bioinformatic analysis. The results from both workflows were interpreted utilizing standardized criteria, which is necessary to avoid reporting nonpathogenic organisms. The RPIP targeted workflow identified AMR markers associated with phenotypic resistance in some bacteria but incorrectly identified

Clostrididioides difficile is associated with adverse clinical outcomes and increased morbidity, mortality, length of hospital stay, and health-care costs.Areas Covered: We searched relevant papers...

Transmission of livestock-associated Staphylococcus aureus clonal complex 9 (LA-SA CC9) between pigs raised on industrial hog operations (IHOs) and humans in the United States is poorly understood. We analyzed whole-genome sequences from 32 international S. aureus CC9 isolates and 49 LA-SA CC9 isolates from IHO pigs and humans who work on or live near IHOs in 10 pig-producing counties in North Carolina, USA. Bioinformatic analysis of sequence data from the 81 isolates demonstrated 3 major LA-SA CC9 clades. North Carolina isolates all fell within a single clade (C3). High-resolution phylogenetic analysis of C3 revealed 2 subclades of intermingled IHO pig and human isolates differing by 0–34 single-nucleotide polymorphisms. Our findings suggest that LA-SA CC9 from pigs and humans share a common source and provide evidence of transmission of antimicrobial-resistant LA-SA CC9 between IHO pigs and humans who work on or live near IHOs in North Carolina.

The fungal pathogen Pneumocystis jirovecii causes Pneumocystis pneumonia. Although the mitochondrial large subunit rRNA gene (mtLSU) is commonly used as a PCR target, a mitochondrial small subunit rRNA gene (mtSSU)–targeted MultiCode PCR assay was developed on the fully automated ARIES platform for detection of P. jirovecii in bronchoalveolar lavage fluid specimens in 2.5 hours. The assay showed a limit of detection of 800 copies/mL (approximately equal to 22 organisms/mL), with no cross-reactivity with other respiratory pathogens. Compared with the reference Pneumocystis-specific direct fluorescent antibody assay (DFA) and mtLSU-targeted PCR assay, the new assay demonstrated sensitivity of 96.9% (31/32) and specificity of 94.6% (139/147) in detecting P. jirovecii in 180 clinical bronchoalveolar lavage fluid specimens. This assay was concordant with all DFA-positive samples and all but one mtLSU PCR-positive sample, and detected eight positive samples that were negative by DFA and mtLSU PCR. Receiver operating characteristic curve analysis revealed an area under the curve of 0.98 and a threshold cycle (CT) cutoff of 39.1 with sensitivity of 90.9% and specificity of 99.3%. The detection of 39.1

Endemic species of coronavirus (HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1) are frequent causes of upper respiratory tract infections. Three highly pathogenic coronaviruses have been associated with outbreaks and epidemics and have challenged clinical microbiology laboratories to quickly develop assays for diagnosis. Their initial characterization was achieved by molecular methods. With the great advance in metagenomic whole-genome sequencing directly from clinical specimens, diagnosis of novel coronaviruses could be quickly implemented into the workflow of managing cases of pneumonia of unknown cause, which will markedly affect the time of the initial characterization and accelerate the initiation of outbreak control measures.

Background Fever of unknown origin (FUO) investigations yield a substantial number of patients with infectious diseases. This systematic review and meta-analysis aimed to quantify more common FUO infectious diseases etiologies and to underscore geographic variation. Methods Four databases (PubMed, Embase, Scopus, and Web of Science) were searched for prospective studies reporting FUO rates among adult patients from 1 January 1997 to 31 March 2021. The pooled proportion for infectious diseases etiology was estimated using the random-effects meta-analysis model. Results Nineteen prospective studies were included with 2667 total cases. No studies were available for Africa or the Americas. Overall, 37.0% (95.0% confidence interval [CI], 30.0%–44.0%) of FUO patients had an infectious disease etiology. Infections were more likely from Southeastern Asia (pooled proportion, 0.49 [95% CI, .43–.55]) than from Europe (pooled proportion, 0.31 [95% CI, .22–.41]). Among specifically reported infectious diseases (n = 832), Mycobacterium tuberculosis complex predominated across all geographic regions (n = 285 [34.3%]), followed by brucellosis (n = 81 [9.7%]), endocarditis (n = 62 [7.5%]), abscesses (n = 61 [7.3%]), herpesvirus (eg, cytomegalovirus and Epstein-Barr virus) infections (n = 60 [7.2%]), pneumonia (n = 54 [6.5%]), urinary tract infections (n = 54 [6.5%]), and enteric fever (n = 40 [4.8%]). Conclusions FUO patients from Southeastern Asia were more likely to have an infectious diseases etiology when compared to other regions. The predominant factor for this finding appears to be differences in disease prevalence among various geographical locations or other factors such as access to timely care and diagnosis. Noting epidemiological disease factors in FUO investigations could improve diagnostic yields and clinical outcomes.

There has been significant progress in detection of bloodstream pathogens in recent decades with the development of more sensitive automated blood culture detection systems and the availability of rapid molecular tests for faster organism identification and detection of resistance genes. However, most blood cultures in clinical practice do not grow organisms, suggesting that suboptimal blood culture collection practices (e.g., suboptimal blood volume) or suboptimal selection of patients to culture (i.e., blood cultures ordered for patients with low likelihood of bacteremia) may be occurring. A national blood culture utilization benchmark does not exist, nor do specific guidelines on when blood cultures are appropriate or when blood cultures are of low value and waste resources. Studies evaluating the potential harm associated with excessive blood cultures have focused on blood culture contamination, which has been associated with significant increases in health care costs and negative consequences for patients related to exposure to unnecessary antibiotics and additional testing. Optimizing blood culture performance is important to ensure bloodstream infections (BSIs) are diagnosed while minimizing adverse events from overuse. In this review, we discuss key factors that influence blood culture performance, with a focus on the preanalytical phase, including technical aspects of the blood culture collection process and blood culture indications. We highlight areas for improvement and make recommendations to improve current blood culture practices among hospitalized patients.

Despite improvements in hospital infection prevention and control, healthcare associated infections (HAIs) remain a challenge with significant patient morbidity, mortality, and cost for the healthcare system. In this review, we use a One Health framework (human, animal, and environmental health) to explain the epidemiology, demonstrate key knowledge gaps in infection prevention policy, and explore improvements to control Gram-positive pathogens in the healthcare environment. We discuss patient and healthcare worker interactions with the hospital environment that can lead to transmission of the most common Gram-positive hospital pathogens – methicillin-resistant Staphylococcus aureus, Clostridioides (Clostridium) difficile, and vancomycin-resistant Enterococcus – and detail interventions that target these two One Health domains. We discuss the role of animals in the healthcare settings, knowledge gaps regarding their role in pathogen transmission, and the absence of infection risk mitigation strategies targeting animals. We advocate for novel infection prevention and control programs, founded on the pillars of One Health, to reduce Gram-positive hospital-associated pathogen transmission.

Objectives Traditional antimicrobial susceptibility testing (AST) is growth dependent and time-consuming. With rising rates of drug-resistant infections, a novel diagnostic method is critically needed that can rapidly reveal a pathogen’s antimicrobial susceptibility to guide appropriate treatment. Recently, RNA sequencing has been identified as a powerful diagnostic tool to explore transcriptional gene expression and improve AST. Methods RNA sequencing was used to investigate the potential of RNA markers for rapid molecular AST using Klebsiella pneumoniae and ciprofloxacin as a model. Downstream bioinformatic analysis was applied for optimal marker selection. Further validation on 11 more isolates of K. pneumoniae was performed using quantitative real-time PCR. Results From RNA sequencing, we identified RNA signatures that were induced or suppressed following exposure to ciprofloxacin. Significant shifts at the transcript level were observed as early as 10 min after antibiotic exposure. Lastly, we confirmed marker expression profiles with concordant MIC results from traditional culture-based AST and validated across 11 K. pneumoniae isolates. recA, coaA and metN transcripts harbour the most sensitive susceptibility information and were selected as our top markers. Conclusions Our results suggest that RNA signature is a promising approach to AST development, resulting in faster clinical diagnosis and treatment of infectious disease. This approach is potentially applicable in other models including other pathogens exposed to different classes of antibiotics.

Guidance regarding indications for initial or follow-up blood cultures is limited. We conducted a scoping review of articles published between January 2004 and June 2019 that reported the yield of blood cultures and/or their impact in the clinical management of fever and common infectious syndromes in nonneutropenic adult inpatients. A total of 2893 articles were screened; 50 were included. Based on the reported incidence of bacteremia, syndromes were categorized into low, moderate, and high pretest probability of bacteremia. Routine blood cultures are recommended in syndromes with a high likelihood of bacteremia (eg, endovascular infections) and those with moderate likelihood when cultures from the primary source of infection are unavailable or when prompt initiation of antibiotics is needed prior to obtaining primary source cultures. In syndromes where blood cultures are low-yield, blood cultures can be considered for patients at risk of adverse events if a bacteremia is missed (eg, patient with pacemaker and severe purulent cellulitis). If a patient has adequate source control and risk factors or concern for endovascular infection are not present, most streptococci or Enterobacterales bacteremias do not require routine follow-up blood cultures.

The optimal timing of blood culture (BCx) sets collection has not been evaluated with continuous BCx detection systems. The yield of BCx was similar between short intervals (median, 3 minutes) and longer intervals (median, 16 or 43 minutes) among 5,856 BCx, except for improved polymicrobial bacteremia detection with long-interval BCx.

This quality improvement project aimed to increase IDC for SAB events at a quaternary pediatric medical center. First, we evaluated the local practices regarding pediatric SAB and engaged stakeholders (July 2018-August 2020). We added an advisory comment supporting IDC for SAB to all blood culture results in September 2020. Using statistical process control charts, we monitored the number of SAB events with IDC before a SAB event without IDC. Finally, we evaluated SAB recurrences before and after initiating the advisory comment.In the baseline period, 30 of 49 (61%) SAB events received an IDC with a mean of 1.4 SAB events with IDC before a SAB event without IDC. Postintervention, 22 of 23 (96%) SAB events received IDC with a mean of 14 events with IDC before 1 event without IDC. The SAB recurrence rate was 8%, with 6 events in 4 children; none of the index cases resulting in recurrence received an IDC (An electronic advisory comment supporting IDC for SAB significantly increased the rate of pediatric IDC with no further SAB recurrence episodes following intervention. This low-resource intervention may be considered in other pediatric centers to optimize SAB management.

The automated plate assessment system (APAS Independence; Clever Culture System, Bäch, Switzerland) is an automated imaging station linked with interpretive software that detects target colonies of interest on chromogenic media and sorts samples as negative or presumptive positive. We evaluated the accuracy of the APAS to triage methicillin-resistant Staphylococcus aureus (MRSA) and S. aureus cultures using chromogenic medium compared to that by human interpretation. Patient samples collected from the nares on ESwabs were plated onto BD BBL CHROMagar MRSA II and BD BBL CHROMagar Staph aureus and allowed to incubate for 20 to 24 h at 37°C in a non-CO2 incubator. Mauve colonies are suggestive of S. aureus and were confirmed with latex agglutination. Following incubation, samples were first interrogated by APAS before being read by a trained technologist blinded to the APAS interpretation. The triaging by both APAS and the technologists was compared for accuracy. Any discordant results required further analysis by a third reader. Over a 5-month period, 5,913 CHROMagar MRSA cultures were evaluated. Of those, 236 were read as concordantly positive, 5,525 were read as concordantly negative, and 152 required discordant analysis. Positive and negative percent agreements (PPA and NPA, respectively) were 100% and 97.3%, respectively. The APAS detected 5 positive cultures that were missed by manual reading and determined to be true positives. In a separate analysis, 744 CHROMagar Staph aureus plates were read in parallel. Of these, 133 were concordantly positive, 585 were concordantly negative, and 26 required discordant analysis. PPA and NPA were 95.7% and 96.7%, respectively. This study confirmed the high sensitivity of digital image analysis by the APAS Independence such that negative cultures can be reliably reported without technologist intervention (negative predictive values [NPVs] of 100% for CHROMagar MRSA and 99.0% for CHROMagar Staph aureus). Triaging using the APAS Independence may provide great efficiency in a laboratory with high throughput of MRSA and S. aureus surveillance cultures.

The global threat of antibiotic resistance underscores critical but unmet needs for rapid antibiotic susceptibility testing (AST) technologies. To this end, droplet microfluidic-based single-cell AST offers promise by achieving unprecedented rapidity, but its potential for clinical use is marred by the capacity of testing one to few antibiotic conditions per device, which falls short from the required scale in clinically relevant scenarios. To lift the scalability constraint in rapid single-cell AST technologies, a new cascaded droplet microfluidic platform that can streamline bacteria/antibiotic mixing, single-cell encapsulation within picoliter droplets, incubation, and detection in a continuous, assembly-line-like workflow is developed. The scalability of the platform is demonstrated by generating 32 groups of ≈10 000 droplets with custom antibiotic conditions within a single device, from which a new statistics-based method is used to analyze the single cell data and produce clinically useful antibiograms with minimum inhibitory concentrations in ≈90 min for the first antibiotic, plus 2 min for each subsequent antibiotic condition. Potential clinical utility of this platform is demonstrated by testing three clinical isolates and eight urine specimens against four frequently used antibiotics, and 100% and 93.8% categorical agreements are achieved compared to laboratory-based results that became available after 48 h.

OBJECTIVEVentricular shunt infection remains an issue leading to high patient morbidity and cost, warranting further investigation. The authors sought to create an animal model of shunt infection that could be used to evaluate possible catheter modifications and innovations.METHODSThree dogs underwent bilateral ventricular catheter implantation and inoculation with methicillin-sensitive Staphylococcus aureus (S. aureus). In 2 experimental animals, the catheters were modified with a polymer containing chemical “pockets” loaded with vancomycin. In 1 control animal, the catheters were polymer coated but without antibiotics. Animals were monitored for 9 to 11 days, after which the shunts were explanted. MRI was performed after shunt implantation and prior to catheter harvest. The catheters were sonicated prior to microbiological culture and also evaluated by electron microscopy. The animals’ brains were evaluated for histopathology.RESULTSAll animals underwent successful catheter implantation. The animals developed superficial wound infections, but no neurological deficits. Imaging demonstrated ventriculitis and cerebral edema. Harvested catheters from the control animal demonstrated > 104 colony-forming units (CFUs) of S. aureus. In the first experimental animal, one shunt demonstrated > 104 CFUs of S. aureus, but the other demonstrated no growth. In the second experimental animal, one catheter demonstrated no growth, and the other grew trace S. aureus. Brain histopathology revealed acute inflammation and ventriculitis in all animals, which was more severe in the control.CONCLUSIONSThe authors evaluated an animal model of ventricular shunting and reliably induced features of shunt infection that could be microbiologically quantified. With this model, investigation of pathophysiological and imaging correlates of infection and potentially beneficial shunt catheter modifications is possible.

Background Long-term effects of chronic and/or recurrent C. difficile infections (CDI) are not well understood, and any potential role of CDI in colorectal cancer (CRC) risk is presently unknown. While pursuing efforts to identify novel procarcinogenic microbes, we identified two mucosal slurries from CRC patients (3728T and 3752T) that were tumorigenic in germ-free (GF) ApcMin/+ mice. Surprisingly, both of these CRC patient slurries were positive for C. difficile by 16S rRNA amplicon sequencing. Given the ability of other chronic infections to promote tumorigenesis (e.g., H. pylori), we hypothesized that chronic colonization with C. difficile could promote tumorigenesis in the colon. Methods A consortium of 30 bacterial isolates including a toxigenic tcdA+ tcdB+ C. difficile strain (CIm_3728T) was cultured from GF ApcMin/+ mice gavaged with the 3728T slurry. This consortium was gavaged into additional GF ApcMin/+ mice with or without C. difficile strains CIm_3728T, CIm_3752T (isolated from mice gavaged with the 3752T slurry), or isogenic tcdA/tcdB mutants of the M7404 R027 strain. Single cell RNA sequencing (scRNAseq), high dimensional (HD) flow cytometry, and fluorescence in situ hybridization (FISH) with EUB338 and Cd198 probes were performed on distal colons from mice gavaged with either complex CRC slurries or the 3728T isolates with CIm_3728T. Results C. difficile strains drove tumorigenesis of the 3728T isolate mixture (Fig. 1A,B). Tumorigenesis was associated with early procarcinogenic signaling and spatial changes including induction of Wnt signaling in colonic epithelial progenitor cells by scRNAseq, IL-17 induction in immune cells by HD flow cytometry, and bacterial biofilm invasion deep into epithelial crypts by FISH. Tumorigenesis correlated with chronic colonization with toxigenic strains of C. difficile and was toxin-dependent, as toxin mutant strains (M7404 tcdA-tcdB-) did not induce tumors. Figure 1. C. difficile strains from CRC patients induce distal colonic tumorigenesis in germ-free (GF) ApcMin/+ mice. A consortium of 30 bacteria, including C. difficile, were isolated from mice gavaged with the 3728T human CRC mucosal slurry. These isolates were then gavaged into additional GF ApcMin/+ mice, with or without C. difficile isolates from mice gavaged with the 3728T slurry or 3752T slurry. (A) Colonic tumor numbers in GF ApcMin/+ mice at 10 wk p.i. demonstrate that C. difficile (Cd) drives the tumorigenesis of this 30-member bacterial consortium. (B) Gross tumors can be observed in the colon of a representative mouse gavaged with the 3728T isolates with the CIm_3728T (top) or CIm_3752T (middle) strain of C. difficile but not in a mouse gavaged with the isolates lacking C. difficile (bottom). Conclusion Toxigenic C. difficile strains isolated from human CRC mucosal slurries were pro-carcinogenic in mice, suggesting that C. difficile is a potential driver of CRC. Given the public health burden of C. difficile, further studies are warranted to determine whether C. difficile infections (initial, recurrent, and chronic asymptomatic) increase CRC risk in patients. Disclosures Jada Domingue, PhD, AstraZeneca (Employee) James White, PhD, Personal Genome Diagnostics (Consultant) Patricia J. Simner, PhD, Accelerate Diagnostics (Grant/Research Support)Affinity Biosensors (Grant/Research Support)BD Diagnostics (Consultant, Grant/Research Support)GeneCapture (Consultant)OpGen, Inc (Consultant, Grant/Research Support)Shionogi, Inc (Consultant) Karen C. Carroll, MD, MeMed (Scientific Research Study Investigator)Meridian Diagnostics, Inc. (Grant/Research Support)Pattern Diagnostics (Advisor or Review Panel member)Scanogen, Inc. (Advisor or Review Panel member) Karen C. Carroll, MD, Pattern Diagnostics, Inc. (Individual(s) Involved: Self): Grant/Research Support; Scanogen, Inc. (Individual(s) Involved: Self): Consultant Cynthia L. Sears, MD, Bristol Myers Squibb (Grant/Research Support)Ferring (Advisor or Review Panel member)Janssen (Grant/Research Support)

The rise of highly transmissible SARS-CoV-2 variants brings new challenges and concerns with vaccine efficacy, diagnostic sensitivity, and public health responses in the fight to end the pandemic. Widespread detection of variant strains will be critical to inform policy decisions to mitigate further spread, and post-pandemic multiplexed screening of respiratory viruses will be necessary to properly manage patients presenting with similar respiratory symptoms. In this work, we have developed a portable, magnetofluidic cartridge platform for automated PCR testing in n = 116) with an overall sensitivity/specificity of 98.1%/95.2%, 85.7%/100%, 100%/98.2%, respectively, for SARS-CoV-2, Influenza A, and Influenza B. Further testing with saliva (n = 14) demonstrated successful detection of all SARS-CoV-2 positive samples with no false-positives.

The signal-to-cutoff (S/CO) ratio of the HIV antigen/antibody test may help immediately to differentiate true-positive results from false-positive results, which may be particularly useful in time-sensitive circumstances, such as when providing emergency department (ED) care.Seven US EDs with HIV screening programs using HIV antigen/antibody assays.This cross-sectional study of existing data correlated S/CO ratios with confirmed HIV status. Test characteristics at predetermined S/CO ratios and the S/CO ratio with the best performance by receiver operator characteristic (ROC) curve were calculated.Of 1035 patients with a reactive HIV antigen/antibody test, 232 (22.4%) were confirmed HIV-negative and 803 (77.6%) were confirmed HIV-positive. Of the 803 patients, 713 (88.8%) experienced chronic infections and 90 (11.2%) experienced acute infections. S/CO ratios were greater for HIV-positive (median 539.2) than for HIV-negative patients (median 1.93) (P0.001) and lower for acute infection (median 22.8) than for chronic infection (median 605.7) (P0.001). All patients with an S/CO ratio1.58 (n = 93) were HIV-negative (NPV 100%), and nearly all with an S/CO ≥ 20.7 (n = 760) (optimal level by ROC analysis) were HIV-positive (PPV 98.6%). Of patients with S/CO values between 1.58 and 20.7 (n = 182), 29.7% were HIV-positive.The S/CO ratio may be used in real time to classify most ED patients as almost certain to be either HIV-positive or HIV-negative long before nucleic acid confirmatory testing is available. When combined with clinical judgment, this could guide preliminary result disclosure and management.

This case-control study investigates the association between nasal microbial profiles and odds of infection after elective surgical procedures., Key Points Question Can characterization of microbiota present on preoperative nasal swab estimate risk for postoperative infection in patients undergoing elective surgical procedures? Findings In this case-control study, 167 patients were classified to nasal microbial profile cluster 1 and 30 patients to cluster 2 based on 16S ribosomal RNA gene sequencing. Classification to cluster 2 was associated with statistically significantly higher odds of infection (ie, deep surgical site infection, bacteremia, or pneumonia) after surgical procedure, independent of covariates, including nasal carriage of Staphylococcus aureus on preoperative culture and intrasample microbial diversity (ie, α diversity). Meaning These findings suggest that the nasal microbiome is an independent risk factor associated with infectious outcomes after elective surgical procedures., Importance The association of the nasal microbiome with outcomes in surgical patients is poorly understood. Objective To characterize the composition of nasal microbiota in patients undergoing clean elective surgical procedures and to examine the association between characteristics of preoperative nasal microbiota and occurrence of postoperative infection. Design, Setting, and Participants Using a nested matched case-control design, 53 individuals who developed postoperative infection were matched (approximately 3:1 by age, sex, and surgical procedure) with 144 individuals who were not infected (ie, the control group). The 2 groups were selected from a prospective cohort of patients undergoing surgical procedures at 2 tertiary care university hospitals in Baltimore, Maryland, who were at high risk for postoperative infectious complications. Included individuals were aged 40 years or older; had no history of autoimmune disease, immunocompromised state, immune-modulating medication, or active infection; and were scheduled to undergo elective cardiac, vascular, spinal, or intracranial surgical procedure. Data were analyzed from October 2015 through September 2020. Exposures Nasal microbiome cluster class served as the main exposure. An unsupervised clustering method (ie, grades of membership modeling) was used to classify nasal microbial samples into 2 groups based on features derived from 16S ribosomal RNA gene sequencing. The microbiome cluster groups were derived independently and agnostic of baseline clinical characteristics and infection status. Main Outcomes and Measures Composite of surgical site infection, bacteremia, and pneumonia occurring within 6 months after surgical procedure. Results Among 197 participants (mean [SD] age, 64.1 [10.6] years; 63 [37.7%] women), 553 bacterial taxa were identified from preoperative nasal swab samples. A 2-cluster model (with 167 patients in cluster 1 and 30 patients in cluster 2) accounted for the largest proportion of variance in microbial profiles using grades of membership modeling and was most parsimonious. After adjusting for potential confounders, the probability of assignment to cluster 2 was associated with 6-fold higher odds of infection after surgical procedure (odds ratio [OR], 6.18; 95% CI, 3.33-11.7; P

In the fight against COVID-19, there remains an unmet need for point-of-care (POC) diagnostic testing tools that can rapidly and sensitively detect the causative SARS-CoV-2 virus to control disease transmission and improve patient management. Emerging CRISPR-Cas-assisted SARS-CoV-2 detection assays are viewed as transformative solutions for POC diagnostic testing, but lack of streamlined sample preparation and full integration within an automated and portable device hamper their potential for POC use. We report herein POC-CRISPR – a single-step CRISPR-Cas-assisted assay that leverages facile magnetic concentration and transport of nucleic acid-binding magnetic beads to incorporate sample preparation with minimal manual operation. Moreover, POC-CRISPR has been adapted into a compact thermoplastic cartridge within a palm-sized yet fully-integrated and automated device. During analytical evaluation, POC-CRISPR was able detect 1 genome equivalent/μL SARS-CoV-2 RNA from a sample volume of 100 μL in < 30 min. Finally, when evaluated with 27 unprocessed clinical nasopharyngeal swab eluates that were pre-typed by standard RT-qPCR (Cq values ranged from 18.3 to 30.2 for the positive samples), POC-CRISPR achieved 27 out of 27 concordance and could detect positive samples with high SARS-CoV-2 loads (Cq < 25) in 20 min.

With the approach of respiratory virus season in the Northern Hemisphere, clinical microbiology and public health laboratories will need rapid diagnostic assays to distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza virus and respiratory syncytial virus (RSV) infections for diagnosis and surveillance. In this study, the clinical performance of the Xpert Xpress SARS-CoV-2/Flu/RSV test (Cepheid, Sunnyvale, CA, USA) for nasopharyngeal swab specimens was evaluated in four centers: Johns Hopkins Medical Microbiology Laboratory, Northwell Health Laboratories, NYC Public Health Laboratory, and Los Angeles County/University of Southern California (LAC+USC) Medical Center., With the approach of respiratory virus season in the Northern Hemisphere, clinical microbiology and public health laboratories will need rapid diagnostic assays to distinguish severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from influenza virus and respiratory syncytial virus (RSV) infections for diagnosis and surveillance. In this study, the clinical performance of the Xpert Xpress SARS-CoV-2/Flu/RSV test (Cepheid, Sunnyvale, CA, USA) for nasopharyngeal swab specimens was evaluated in four centers: Johns Hopkins Medical Microbiology Laboratory, Northwell Health Laboratories, NYC Public Health Laboratory, and Los Angeles County/University of Southern California (LAC+USC) Medical Center. A total of 319 nasopharyngeal swab specimens, positive for SARS-CoV-2 (n = 75), influenza A virus (n = 65), influenza B virus (n = 50), or RSV (n = 38) or negative (n = 91) by the standard-of-care nucleic acid amplification tests at each site, were tested using the Cepheid panel test. The overall positive percent agreement for the SARS-CoV-2 target was 98.7% (n = 74/75), and the negative agreement was 100% (n = 91), with all other analytes showing 100% total agreement (n = 153). Standard-of-care tests to which the Cepheid panel was compared included the Cepheid Xpert Xpress SARS-CoV-2, Cepheid Xpert Xpress Flu/RSV, GenMark ePlex respiratory panel, BioFire respiratory panel 2.1 and v1.7, DiaSorin Simplexa COVID-19 Direct, and Hologic Panther Fusion SARS-CoV-2 assays. The Xpert Xpress SARS-CoV-2/Flu/RSV test showed high sensitivity and accuracy for all analytes included in the test. This test will provide a valuable clinical diagnostic and public health solution for detecting and differentiating SARS-CoV-2, influenza A and B virus, and RSV infections during the current respiratory virus season.

Bronchoalveolar lavage (BAL) culture is a standard, though time-consuming, approach for identifying microorganisms in patients with severe lower respiratory tract (LRT) infections. The sensitivity of BAL culture is relatively low, and prior antimicrobial therapy decreases the sensitivity further, leading to overuse of empirical antibiotics. The Unyvero LRT BAL Application (Curetis GmbH, Germany) is a multiplex molecular panel that detects 19 bacteria, 10 antibiotic resistance markers, and a fungus, Pneumocystis jirovecii, in BAL fluid in ∼4.5 h. Its performance was evaluated using 1,016 prospectively collected and 392 archived specimens from 11 clinical trial sites in the United States. Overall positive and negative percent agreements with culture results for identification of bacteria that grow in routine cultures were 93.4% and 98.3%, respectively, with additional potential pathogens identified by Unyvero in 21.7% of prospectively collected specimens. For detection of P. jirovecii, the positive percent agreement with standard testing was 87.5%. Antibiotic resistance marker results were compared to standard antibiotic susceptibility test results to determine positive predictive values (PPVs). PPVs ranged from 80 to 100%, based on the microorganism and specific resistance marker(s). The Unyvero LRT BAL Application provides accurate detection of common agents of bacterial pneumonia and of P. jirovecii. The sensitivity and rapidity of this panel suggest significant clinical value for choosing appropriate antibiotics and for antibiotic stewardship.

The rise of antimicrobial-resistant pathogens can be attributed to the lack of a rapid pathogen identification (ID) or antimicrobial susceptibility testing (AST), resulting in delayed therapeutic decisions at the point of care. Gonorrhea is usually empirically treated, with no AST results available before treatment, thus contributing to the rapid rise in drug resistance. Here, we present a rapid AST platform using RNA signatures for

Therapy animals in hospital animal-assisted intervention programs are an invaluable part of holistic patient care. However, therapy dogs may be exposed to hospital-associated pathogens through these activities. This pilot study sought to examine the effect of topical chlorhexidine application, used as an infection control measure, on the microbial composition of the skin and mucous membranes of therapy dogs. We found that the chlorhexidine decolonization intervention altered microbial alpha diversity and shifted microbial structures in these therapy dogs, and particularly influenced more phylogenetically rare taxa. Specifically, the intervention reduced the abundance of Staphylococcus pseudintermedius, the archetypal canine commensal and a frequent cause of opportunistic infections. However, it did not reduce levels of S. aureus, which is a common hospital-associated pathogen of people. These preliminary findings highlight the importance of considering holistic microbial communities when undertaking infection control strategies, and stress the need for further research to understand the unintended consequences of antiseptic use on therapy dogs.

BackgroundMicrobial sharing between humans and animals has been demonstrated in a variety of settings. However, the extent of microbial sharing that occurs within the healthcare setting during animal-assisted intervention programs, a validated and valuable part of holistic patient wellness, is unknown. Understanding microbial transmission between patients and therapy animals can provide important insights into potential health benefits for patients, in addition to addressing concerns regarding potential pathogen transmission that limits program utilization. This study evaluated the potential for microbial sharing between pediatric patients and therapy dogs, and tested whether patient-dog contact level and a dog decolonization protocol modified this sharing.Methods and ResultsPatients, therapy animals, and the hospital environment were sampled before and after every group therapy session and samples underwent 16S rRNA sequencing to characterize microbial communities. Both patients and animals experienced changes in the relative abundance and overall diversity of their nasal microbiome, suggesting that exchange of microorganisms had occurred. Increased contact was associated with greater sharing between patients and therapy animals, as well as between patients. A topical chlorhexidine-based dog decolonization intervention was associated with decreased microbial sharing between therapy dogs and patients, particularly from the removal of rarer microbiota from the dog, but did not significantly affect sharing between patients.ConclusionThese data suggest that the therapy animal is both a potential source of and a vehicle for the transfer of microorganisms to patients but not necessarily the only source. The relative contribution of other potential sources (e.g., other patients, the hospital environment) should be further explored to determine their relative importance.