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Several studies have reported the coexistence of gastric gastrointestinal stromal tumors (GISTs) with many primary carcinomas such as gastric and renal cell carcinomas. However, to date reports about the coexistence of gastric GISTs and colorectal adenocarcinoma are limited. Herein we report a unique case of gastric GIST coexisting synchronously with rectal adenocarcinoma in a 36-year-old male patient who presented with weight loss, vomiting, and bleeding per rectum. Computed tomography (CT) revealed circumferential rectal mass coexistent with an irregular gastric soft tissue mass. The diagnosis of rectal adenocarcinoma and gastric GIST was established by immunohistological evaluation of the colonoscopic (rectum) and CT-guided (stomach) biopsies. The patient received concomitant chemoradiotherapy for the rectal adenocarcinoma and neoadjuvant imatinib for the gastric GIST. This was followed by low anterior resection with total mesorectal excision and wedge resection of the gastric mass. Follow-up of the patient for 1.5 years revealed no evidence of disease recurrence. We also present a minireview of the literature that provides insights into this subject as a separate section.

Background Leptospira can cause severe infections but their diagnosis can be challenging. Diagnosis is limited by nonspecific, protean clinical symptoms; the fastidious nature of Leptospira; and poor specificity, sensitivity, and long turnaround times of serologic testing. Given these limitations, identifying epidemiological clusters of leptospirosis can be challenging. Rapid, non-invasive diagnosis of leptospirosis by microbial cell-free DNA (mcfDNA) next-generation sequencing (NGS) of plasma offers a means to overcome these limitations in individual diagnosis and public health epidemiological surveillance. Methods The Karius Test® (KT) detects and quantifies mcfDNA in molecules/µL (MPM) in plasma from a curated genomic database of >20,000 organisms reporting on >1000 pathogens through the Karius CLIA certified/CAP accredited lab (Redwood City, CA). Four KT detections of Leptospira santarosai were identified April - August 2021 across four independent United States institutions. Clinical review was performed by pediatric infectious diseases consultants. Results KT detected L. santarosai in four adolescents with travel to Costa Rica or Belize (Table). A broad work-up was performed in all patients. McfDNA NGS was the first test to identify L. santarosai as the diagnosis in all cases with an average time to diagnosis of 2.5+/- 0.58 days from sample collection, enabling narrowed, targeted antibiotic treatment. L. santarosai mcfDNA was positive in three patients despite pretreatment; average L. santarosai mcfDNA concentration was 173.5+/- 186.1 MPM. Leptospira serologies were confirmatory in three patients, potential epidemiological exposures were identified in all patients, and all recovered. Conclusion KT enabled rapid, non-invasive diagnosis of diverse manifestations of L. santarosai against a competing broad differential in four adolescent travelers returning from Costa Rica or Belize. The resolution of the etiology of this cluster of patients with leptospirosis demonstrates the potential utility of mcfDNA to quickly diagnose and support case definitions of reportable diseases and the potential importance of plasma mcfDNA NGS in public health and epidemiological surveillance. Disclosures Asim A. Ahmed, MD, Karius: Employee|Karius: Stocks/Bonds Sara Saporta-Keating, MS, MD, Pfizer: Co-investigator in investigator-driven study sponsored by Pfizer.

Background Pneumonia is the most common infectious cause of morbidity and excess mortality complicating hematopoietic cell transplantation (HCT) and treatment of hematologic malignancy. Standard bronchoscopic and noninvasive microbiologic testing identify causative pathogens in less than half of cases. The Karius Test, a plasma next-generation sequencing assay of microbial cell-free DNA, may improve diagnostic yield in these patients. Methods Patients with active hematologic malignancy or recent HCT undergoing bronchoscopy for suspected pneumonia were prospectively enrolled in this observational study conducted at 10 United States medical centers. A panel of expert clinicians blinded to Karius Test results reviewed a standardized panel of microbiologic and molecular testing from bronchoalveolar lavage and blood samples for bacterial and fungal testing, nasopharyngeal swab for respiratory viral testing, imaging results, clinical documentation, and any additional microbiologic or molecular testing collected per usual standard of care to adjudicate a probable cause of pneumonia. The panel then adjudicated whether a probable cause of pneumonia or other clinically relevant infection was identified by the Karius Test. Results Between January 3, 2020 and February 4, 2022, 257 patients were enrolled. A planned interim analysis of the first 69 sequentially enrolled patients in the per protocol population was conducted. An adjudicated probable cause of pneumonia was identified by standard care in 18/69 (26%) patients. The Karius Test identified an adjudicated probable cause of pneumonia in 10/51 (20%) patients when no cause of pneumonia was identified by standard care testing. The combination of standard care and the Karius Test together identified a probable cause of pneumonia in 28/69 (41%) patients. At least one additional pathogen adjudicated as a probable cause of pneumonia was identified by the Karius Test in 6/18 (33%) of patients with positive standard care testing. Conclusion The Karius Test notably increased the probability of identifying a pathogenic cause of pneumonia among immunocompromised patients undergoing bronchoscopy. The additive diagnostic value of the Karius Test may significantly enhance management of this common condition. Disclosures Roy F. Chemaly, MD/MPH, Karius: Advisor/Consultant|Karius: Grant/Research Support Radha Duttagupta, PhD, Karius Inc: Stocks/Bonds Sanjeet S. Dadwal, MD, FACP, FIDSA, AlloVir: Advisor/Consultant|AlloVir: Grant/Research Support|Ansun Biopharma: Grant/Research Support|Aseptiscope: Advisor/Consultant|Aseptiscope: Stocks/Bonds|Astellas: Speaker's Bureau|Cidara: Advisor/Consultant|Gilead: Grant/Research Support|Karius: Grant/Research Support|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Speaker's Bureau|Takeda: Speaker's Bureau Joshua A. Hill, MD, Allovir: Advisor/Consultant|Allovir: Grant/Research Support|Covance/CSL: Advisor/Consultant|CRISPR: Advisor/Consultant|Deverra: Grant/Research Support|Gilead: Grant/Research Support|Karius: Advisor/Consultant|Karius: Grant/Research Support|Merck: Grant/Research Support|Octapharma: Advisor/Consultant|OptumHealth: Advisor/Consultant|Oxford Immunotec: Grant/Research Support|Pfizer: Advisor/Consultant|Symbio: Advisor/Consultant|Takeda: Advisor/Consultant Ghady Haidar, MD, Karius, Allovir, and AstraZeneca: Grant/Research Support Alfred Luk, MD, Karius: Grant/Research Support Jamie Todd, MD, Altavant Sciences: Advisor/Consultant|AstraZeneca: Grant/Research Support|Boehringer Ingelheim: Grant/Research Support|CareDx: Grant/Research Support|Cellarity: Advisor/Consultant|Natera: Advisor/Consultant Genovefa Papanicolaou, MD, AlloVir: Board Member|AlloVir: Serve as member of DSMC|Amplyx: Board Member|Amplyx: Serve as member of DSMC|Astellas: Advisor/Consultant|Cidara: Advisor/Consultant|CSL Behring: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Merck: Investigator for Merck|MSD: Advisor/Consultant|Octapharma: Advisor/Consultant|Octapharma: Board Member|Octapharma: Serve as EAC member|Partners RX: Advisor/Consultant|SymBio: Advisor/Consultant|Takeda: Advisor/Consultant|Takeda: Grant/Research Support|Takeda: Investigator for Takeda|Vera: Board Member|Vera: Serve as member of DSMC Elena Nemirovich-Danchenko, MD PhD, Karius: Stocks/Bonds Mona Mughar, BS, Karius: Stocks/Bonds Sudeb Dalai, MD, Karius: Stocks/Bonds Sudeb Dalai, MD, Karius: Stocks/Bonds Yuen Cho, MS, CLS(CA-DPH), Karius: Stocks/Bonds Asim A. Ahmed, MD, Karius: Employee|Karius: Stocks/Bonds Desiree Hollemon, MSN, MPH, Karius: Stocks/Bonds David K. Hong, MD, Janssen Pharmaceutical Companies of Johnson & Johnson: Employee|Vir Biotechnology: Employee|Vir Biotechnology: Stocks/Bonds Marla Lay Vaughn, BS, MT(ASCP), Karius: Employee|Karius: Stocks/Bonds Tim Blauwkamp, PhD, Karius: Board Member|Karius: Ownership Interest Zivjena Vucetic, MD, Karius: Stocks/Bonds Rina Romano, BS, Karius Inc: Stocks/Bonds|Karius Inc: Stocks/Bonds Rina Romano, BS, Karius Inc: Stocks/Bonds|Karius Inc: Stocks/Bonds Rina Romano, BS, Karius Inc: Stocks/Bonds|Karius Inc: Stocks/Bonds Vance G. Fowler, Jr, MD, MHS, Affinergy: Grant/Research Support|Affinergy: Honoraria|Affinium: Honoraria|Amphliphi Biosciences: Honoraria|ArcBio: Stocks/Bonds|Basilea: Grant/Research Support|Basilea: Honoraria|Bayer: Honoraria|C3J: Honoraria|Cerexa/Forest/Actavis/Allergan: Grant/Research Support|Contrafect: Grant/Research Support|Contrafect: Honoraria|Cubist/Merck: Grant/Research Support|Debiopharm: Grant/Research Support|Deep Blue: Grant/Research Support|Destiny: Honoraria|Genentech: Grant/Research Support|Genentech: Honoraria|Integrated Biotherapeutics: Honoraria|Janssen: Grant/Research Support|Janssen: Honoraria|Karius: Grant/Research Support|Medicines Co.: Honoraria|MedImmune: Grant/Research Support|MedImmune: Honoraria|NIH: Grant/Research Support|Novartis: Grant/Research Support|Novartis: Honoraria|Pfizer: Grant/Research Support|Regeneron: Grant/Research Support|Regeneron: Honoraria|Sepsis diagnostics: Sepsis diagnostics patent pending|UpToDate: Royalties|Valanbio: Stocks/Bonds Thomas L. Holland, MD, Aridis: Advisor/Consultant|Lysovant: Advisor/Consultant.

BackgroundSecondary infection (SI) diagnosis in COVID-19 is challenging, due to overlapping clinical presentations, practical limitations in obtaining samples from the lower respiratory tract (LRT), and low sensitivity of microbiologic cultures.Research QuestionCan metagenomic sequencing of plasma microbial cell-free DNA (mcfDNA-Seq) help diagnose SIs complicating COVID-19?Study Design and MethodsWe enrolled 42 inpatients with COVID-19 classified as microbiologically-confirmed SI (Micro-SI, n=8), clinically-diagnosed SI (Clinical-SI, n=13, i.e. empiric antimicrobials), or no clinical suspicion for SI (No-Suspected-SI, n=21) at time of enrollment. From baseline and follow-up plasma samples (days 5 and 10 post-enrollment), we quantified mcfDNA for all detected microbes by mcfDNA sequencing and measured nine host-response biomarkers. From LRT samples among intubated subjects, we quantified bacterial burden with 16S rRNA gene quantitative PCR.ResultsWe performed mcfDNA-Seq in 82 plasma samples. Sequencing was successful in 60/82 (73.2%) samples, which had significantly lower levels of human cfDNA than failed samples (p10 mcfDNA, p=0.03).InterpretationHigh circulating levels of mcfDNA in a substantial proportion of patients with COVID-19 without clinical suspicion for SI suggest that SIs may often remain undiagnosed. McfDNA-Seq, when clinically available, can offer a non-invasive diagnostic tool for pathogen identification, with prognostic value on host inflammatory response and clinical outcomes.

Background Over 1 million Americans undergo joint replacement each year, and approximately 1 in 75 will incur a periprosthetic joint infection. Effective treatment necessitates pathogen identification, yet standard-of-care cultures fail to detect organisms in 10% to 20% of cases and require invasive sampling. We hypothesized that cell-free DNA (cfDNA) fragments from microorganisms in a periprosthetic joint infection can be found in the bloodstream and utilized to accurately identify pathogens via next-generation sequencing. Methods In this prospective observational study performed at a musculoskeletal specialty hospital in the U.S., we enrolled 53 adults with validated hip or knee periprosthetic joint infections. Participants had peripheral blood drawn immediately prior to surgical treatment. Microbial cfDNA from plasma was sequenced and aligned to a genome database with >1,000 microbial species. Intraoperative tissue and synovial fluid cultures were performed per the standard of care. The primary outcome was accuracy in organism identification with use of blood cfDNA sequencing, as measured by agreement with tissue-culture results. Results Intraoperative and preoperative joint cultures identified an organism in 46 (87%) of 53 patients. Microbial cfDNA sequencing identified the joint pathogen in 35 cases, including 4 of 7 culture-negative cases (57%). Thus, as an adjunct to cultures, cfDNA sequencing increased pathogen detection from 87% to 94%. The median time to species identification for cases with genus-only culture results was 3 days less than standard-of-care methods. Circulating cfDNA sequencing in 14 cases detected additional microorganisms not grown in cultures. At postoperative encounters, cfDNA sequencing demonstrated no detection or reduced levels of the infectious pathogen. Conclusions Microbial cfDNA from pathogens causing local periprosthetic joint infections can be detected in peripheral blood. These circulating biomarkers can be sequenced from noninvasive venipuncture, providing a novel source for joint pathogen identification. Further development as an adjunct to tissue cultures holds promise to increase the number of cases with accurate pathogen identification and improve time-to-speciation. This test may also offer a novel method to monitor infection clearance during the treatment period. Level of evidence Diagnostic Level II. See Instructions for Authors for a complete description of levels of evidence.

His-refractory premature ventricular complexes perturbing a supraventricular tachycardia (SVT) establish the presence of an accessory pathway (AP). Earlier premature ventricular complexes (ErPVCs) may perturb SVTs but are considered nondiagnostic.The purpose of this study was to test the hypothesis that an ErPVC will always show a difference35 ms in its advancement of the next atrial activation during atrioventricular nodal reentrant tachycardia (AVNRT). During atrioventricular reentrant tachycardia (AVRT), a PVC delivered close to the circuit can result in greater advancement of atrial activation due to retrograde conduction via an AP. Thus, an AP response, defined as ErPVC (HSixty-five consecutive patients with SVT were retrospectively evaluated. ErPVCs were defined when the ventricular pacing stimulus was35 ms ahead of the His during tachycardia.Among the 65 cases, 43 were AVNRT and 22 AVRT. Fourteen AVRT cases had an AP response with a mean HAn AP response to PVCs (A

BackgroundDetection and sequencing of circulating microbial cell-free DNA (mcfDNA) in plasma is an increasingly popular tool for diagnosing many infectious diseases, but could also be used to monitor the progress of infection. However, the decay of this microbial cell-free DNA in blood following treatment has not been previously characterized.Case PresentationA 53 year-old male was diagnosed withBartonella quintanabioprosthetic aortic valve endocarditis by sequencing of the mcfDNA in the blood (Karius, Redwood City, CA). We then monitored the kinetics of decay of mcfDNA after parenteral antibiotics and valve resection in this individual. We measured plasma mcfDNA (Karius) in serial samples obtained in the operating room to calculate mcfDNA half-life after valve resection. After four weeks of parenteral antibiotics,BartonellamcfDNA signal decreased by 78%. The signal subsequently rose during operative manipulation of the infected valve but dropped 81-fold over four hours following valve resection. The half-life of mcfDNA between the time shortly following resection of the infected valve and 24 to 48 hours post-operatively was between 35 and 115 minutes. The trend in mcfDNA signal was characterized by rapid and then slower phases of decay within 24 hours, and little change between 24 and 48 hours.ConclusionsThis study is one of the first to characterize decay kinetics of mcfDNA and highlights the potential of monitoring mcfDNA in addressing major challenges in infective endocarditis management, including monitoring the response to therapy, and as an early screen for recurrence.

Background The diagnosis of infective endocarditis (IE) can be difficult, particularly if blood cultures fail to yield a pathogen. This study evaluates the potential utility of microbial cell-free DNA (mcfDNA) as a tool to identify the microbial etiology of IE. Methods Blood samples from patients with suspected IE were serially collected. mcfDNA was extracted from plasma and underwent next-generation sequencing. Reads were aligned against a library containing DNA sequences belonging to >1400 different pathogens. mcfDNA from organisms present above a statistical threshold were reported and quantified in molecules per milliliter (MPM). Additional mcfDNA was collected on each subject every 2–3 days for a total of 7 collections or until discharge. Results Of 30 enrolled patients with suspected IE, 23 had definite IE, 2 had possible IE, and IE was rejected in 5 patients by modified Duke Criteria. Only the 23 patients with definite IE were included for analysis. Both mcfDNA and blood cultures achieved a sensitivity of 87%. The median duration of positivity from antibiotic treatment initiation was estimated to be approximately 38.1 days for mcfDNA versus 3.7 days for blood culture (proportional odds, 2.952; P = .02771), using a semiparametric survival analysis. mcfDNA (log10) levels significantly declined (−0.3 MPM log10 units, 95% credible interval −0.45 to −0.14) after surgical source control was performed (pre- vs postprocedure, posterior probability >0.99). Conclusion mcfDNA accurately identifies the microbial etiology of IE. Sequential mcfDNA levels may ultimately help to individualize therapy by estimating a patient’s burden of infection and response to treatment.

Background Noninvasive diagnostic options are limited for invasive mold infections (IMIs). We evaluated the performance of a plasma microbial cell-free DNA sequencing (mcfDNA-Seq) test for diagnosing pulmonary IMI after hematopoietic cell transplant (HCT). Methods We retrospectively assessed the diagnostic performance of plasma mcfDNA-Seq next-generation sequencing in 114 HCT recipients with pneumonia after HCT who had stored plasma obtained within 14 days of diagnosis of proven/probable Aspergillus IMI (n = 51), proven/probable non-Aspergillus IMI (n = 24), possible IMI (n = 20), and non-IMI controls (n = 19). Sequences were aligned to a database including >400 fungi. Organisms above a fixed significance threshold were reported. Results Among 75 patients with proven/probable pulmonary IMI, mcfDNA-Seq detected ≥1 pathogenic mold in 38 patients (sensitivity, 51% [95% confidence interval {CI}, 39%–62%]). When restricted to samples obtained within 3 days of diagnosis, sensitivity increased to 61%. McfDNA-Seq had higher sensitivity for proven/probable non-Aspergillus IMI (sensitivity, 79% [95% CI, 56%–93%]) compared with Aspergillus IMI (sensitivity, 31% [95% CI, 19%–46%]). McfDNA-Seq also identified non-Aspergillus molds in an additional 7 patients in the Aspergillus subgroup and Aspergillus in 1 patient with possible IMI. Among 19 non-IMI pneumonia controls, mcfDNA-Seq was negative in all samples, suggesting a high specificity (95% CI, 82%–100%) and up to 100% positive predictive value (PPV) with estimated negative predictive values (NPVs) of 81%–99%. The mcfDNA-Seq assay was complementary to serum galactomannan index testing; in combination, they were positive in 84% of individuals with proven/probable pulmonary IMI. Conclusions Noninvasive mcfDNA-Seq had moderate sensitivity and high specificity, NPV, and PPV for pulmonary IMI after HCT, particularly for non-Aspergillus species.

This study sought to determine if atrial fibrillation (AF) ablation can be performed safely without bladder catheterization.Patients undergoing AF ablation often receive bladder catheters. Catheterization is associated with potential complications. The ABCD-AF (Avoiding Bladder Catheters During Atrial Fibrillation) ablation study evaluates the advantages of performing AF ablation without routine catheterization.In this single-center, prospective, randomized controlled trial, 80 patients received bladder catheterization (group A), and 80 patients received only as-needed catheterization (group B). The primary endpoint was a composite of cystitis, urethral injury, hematuria, dysuria, or urinary retention.The mean patient age was 63 ± 13 years, and 33% of patients were female. The primary outcome was reached in 45 patients in group A and 11 patients in group B (p 0.001). Urinary tract infection occurred in 7 patients in group A and 2 patients in group B (p = 0.17). Urinary retention occurred in 12 patients in group A and 5 patients in group B (p = 0.07). Randomization to catheterization carried an odds ratio of 8.1 (95% confidence interval [CI]: 3.7 to 17.5; p 0.001), and male sex carried an odds ratio of 3.8 (95% CI: 1.7 to 8.6; p = 0.001) for the primary endpoint. On subgroup analysis, randomization to undergo catheterization had no association with the primary outcome in female patients but had an odds ratio of 14.6 (95% CI: 5.6 to 38.1; p 0.001) in male patients. In multivariable analysis, sex and catheter status remained independently associated with the primary outcome.Bladder catheterization can be safely avoided in patients undergoing AF ablation and is associated with a significant reduction in adverse outcomes, especially in men.

Metagenomic next-generation sequencing (mNGS), the shotgun sequencing of RNA and DNA from clinical samples, has proved useful for broad-spectrum pathogen detection and the genomic surveillance of viral outbreaks. An additional target enrichment step is generally needed for high-sensitivity pathogen identification in low-titre infections, yet available methods using PCR or capture probes can be limited by high cost, narrow scope of detection, lengthy protocols and/or cross-contamination. Here, we developed metagenomic sequencing with spiked primer enrichment (MSSPE), a method for enriching targeted RNA viral sequences while simultaneously retaining metagenomic sensitivity for other pathogens. We evaluated MSSPE for 14 different viruses, yielding a median tenfold enrichment and mean 47% (±16%) increase in the breadth of genome coverage over mNGS alone. Virus detection using MSSPE arboviral or haemorrhagic fever viral panels was comparable in sensitivity to specific PCR, demonstrating 95% accuracy for the detection of Zika, Ebola, dengue, chikungunya and yellow fever viruses in plasma samples from infected patients. Notably, sequences from re-emerging and/or co-infecting viruses that have not been specifically targeted a priori, including Powassan and Usutu, were successfully enriched using MSSPE. MSSPE is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making this method directly applicable for diagnostic laboratory and field use., This study describes a new method that improves the sensitivity of viral detection compared with next-generation sequencing and enables the detection of emerging flaviviruses not specifically targeted a priori. Metagenomic sequencing with spiked primer enrichment is simple, low cost, fast and deployable on either benchtop or portable nanopore sequencers, making it applicable for diagnostic laboratory and field use.

An 11-month-old male infant with ascending paralysis had an unremarkable initial cerebrospinal fluid (CSF) analysis and imaging. Progressive neurological symptoms resulted in repeated CSF sampling, microscopy, and plasma microbial cell-free DNA next-generation sequencing analysis, that in combination with epidemiology, confirmed the diagnosis.

Meningitis and encephalitis are leading causes of central nervous system (CNS) disease and often result in severe neurological compromise or death. Traditional diagnostic workflows largely rely on pathogen-specific tests, sometimes over days to weeks, whereas metagenomic next-generation sequencing (mNGS) profiles all nucleic acid in a sample. In this single-center, prospective study, 68 hospitalized patients with known (n = 44) or suspected (n = 24) CNS infections underwent mNGS from RNA and DNA to identify potential pathogens and also targeted sequencing of viruses using hybrid capture. Using a computational metagenomic classification pipeline based on KrakenUniq and BLAST, we detected pathogen nucleic acid in cerebrospinal fluid (CSF) from 22 subjects, 3 of whom had no clinical diagnosis by routine workup. Among subjects diagnosed with infection by serology and/or peripheral samples, we demonstrated the utility of mNGS to detect pathogen nucleic acid in CSF, importantly for the Ixodes scapularis tick-borne pathogens Powassan virus, Borrelia burgdorferi, and Anaplasma phagocytophilum. We also evaluated two methods to enhance the detection of viral nucleic acid, hybrid capture and methylated DNA depletion. Hybrid capture nearly universally increased viral read recovery. Although results for methylated DNA depletion were mixed, it allowed the detection of varicella-zoster virus DNA in two samples that were negative by standard mNGS. Overall, mNGS is a promising approach that can test for multiple pathogens simultaneously, with efficacy similar to that of pathogen-specific tests, and can uncover geographically relevant infectious CNS disease, such as tick-borne infections in New England. With further laboratory and computational enhancements, mNGS may become a mainstay of workup for encephalitis and meningitis. IMPORTANCE Meningitis and encephalitis are leading global causes of central nervous system (CNS) disability and mortality. Current diagnostic workflows remain inefficient, requiring costly pathogen-specific assays and sometimes invasive surgical procedures. Despite intensive diagnostic efforts, 40 to 60% of people with meningitis or encephalitis have no clear cause of CNS disease identified. As diagnostic uncertainty often leads to costly inappropriate therapies, the need for novel pathogen detection methods is paramount. Metagenomic next-generation sequencing (mNGS) offers the unique opportunity to circumvent these challenges using unbiased laboratory and computational methods. Here, we performed comprehensive mNGS from 68 prospectively enrolled patients with known (n = 44) or suspected (n = 24) CNS viral infection from a single center in New England and evaluated enhanced methods to improve the detection of CNS pathogens, including those not traditionally identified in the CNS by nucleic acid detection. Overall, our work helps elucidate how mNGS can become integrated into the diagnostic toolkit for CNS infections.

Rationale: Severe COVID-19 pneumonia can be complicated by secondary bacterial or fungal infections, but their clinical distinction from isolated SARS-CoV-2 infection is challenging, especially with the more restricted practices regarding invasive diagnostics in patients with COVID-19. We sought to comprehensively screen for secondary infections by DNA pathogens (bacterial, fungal or viral) with a non-invasive, culture-independent metagenomic approach (microbial cell-free DNA sequencing-mcfDNA-Seq), and also examine for the biologic impact of circulating mcfDNA on the host response in COVID-19. Methods: We prospectively enrolled 42 hospitalized patients with COVID-19 and compared them with a historical cohort of mechanically-ventilated patients with culture-positive (n=27) vs. culture-negative pneumonia (n=40) or no clinical infection (n=16 controls). From plasma samples, we performed mcfDNA-Seq with the Karius test (Karius, Inc) and measured 10 host-response biomarkers of innate immunity and epithelial/endothelial injury (IL-6, IL-8, IL-10, RAGE, TNFR1, Angiopoietin-2, Procalcitonin, Fractalkine, Pentraxin-3, ST2). We compared mcfDNA-Seq between clinical groups and examined associations of mcfDNA and biomarker levels with linear regression models. Results: McfDNA-Seq was successful in 33/42 (79%) baseline samples from patients with COVID-19, with nine samples failing QC requirements. McfDNA was detectable in 21/33 (64%) of COVID-19 samples, a proportion significantly lower to culture-positive pneumonia (96%), higher than uninfected controls (31%) and similar to culture-negative pneumonia (56%) (between-groups Fisher's exact p

Background Capnocytophaga canimorsus (Cc) and Pasteurella multocida (Pm) are gram negative bacterial commensal pathogens typically from dogs or cats that can cause severe infection in humans when spread through licks, scratches or bites. The diagnosis of these infections can be limited by: (1) their fastidious nature and difficulty to culture; (2) the nonspecific manifestations of the infections; and (3) the unreliability of dog or cat exposure history. Open-ended microbial cell free DNA (mcfDNA) next-generation sequencing (NGS) offers a potential solution to overcome these limitations. Methods The Karius TestTM (KT) developed and validated in Karius’s CLIA certified/CAP accredited lab in Redwood City, CA detects mcfDNA in plasma. After mcfDNA is extracted and NGS performed, human reads are removed, and remaining sequences are aligned to a curated database of > 1500 organisms. McfDNA from organisms present above a statistical threshold are reported and quantified in molecules/µL (MPM). KT detections of Cc and Pm were reviewed from August 2017 - present; clinical information was obtained with test requisition or consultation upon result reporting. Results KT detected 5 cases of Cc (25,039 MPM +/- 41,062) and 8 cases of Pm (33,264 MPM +/- 69,301) (Table 1). All detections of Cc were in adults (60% male) and included 2 cases of culture-negative endocarditis (one with known liver disease) and one case of sepsis with diffuse rash. Pm detections occurred in 6 adults and 2 children (75% male) and included 2 cases of culture-negative endocarditis, and single cases each of endovascular graft infection, pneumonia, fever of unknown origin, and a cranial dog bite complicated by an abscess. Two patients had immunocompromising conditions including neuroblastoma and aplastic anemia. Conclusion Unbiased, plasma-based mcfDNA NGS provides a rapid, non-invasive test to diagnose diverse clinical infections by Cc and Pm. These cases highlight the potential of the KT to diagnose infections caused by fastidious/unculturable pathogens with non-specific clinical manifestations and broad differential diagnoses. Disclosures Nicholas R. Degner, MD, MPH, MS, Karius Inc. (Employee, Shareholder) Ricardo Galvan-Castillo, MD, Karius Inc. (Employee, Shareholder) Jose Alexander, MD, D(ABMM), FCCM, CIC, SM, MB(ASCP), BCMAS, Karius (Employee) Aparna Arun, MD, Karius (Employee) Ann Macintyre, DO, Karius, Inc. (Employee) Bradley Perkins, MD, Karius, Inc. (Employee) Asim A. Ahmed, MD, Karius, Inc. (Employee) Matthew Smollin, PharmD, Karius, Inc. (Employee)

Background Human herpesvirus 6 (HHV6) has been classified in two distinct variants, HHV6A and HHV6B. Although distinct epidemiology, disease association, biological and immunological properties, their genomes are 90% homologous. Less is known about HHV6A which is typically asymptomatic but can cause severe infection in patients with neurological disorders or HIV. HHV6B infection occurs during childhood, but can reactivate after solid organ and stem cell transplantation. Antibody assays may indicate previous, recent or current infection. Quantitative PCR cannot differentiate active from latent infections and some available qualitative PCR assays are unable to differentiate the two variants. A single open-ended test through plasma-based microbial cell-free DNA (mcfDNA) metagenomic next-generation sequencing (NGS) may overcome these limitations. Methods Karius TestTM (KT) developed and validated in Karius’s CLIA certified/CAP accredited lab, detects mcfDNA from plasma. mcfDNA is extracted, NGS performed, human sequences removed and remaining sequences aligned to a curated pathogen database of > 1500 organisms. Organisms present above a statistical threshold are reported and quantified. For > 85% of tests the time to result reporting is the next day from sample receipt. KT results were reviewed from November-2018 to May-2021 for detections of HHV6A and HHV6B. The comparative incidence of HHV6A and HHVB detections, their age distributions and their quantitative viral concentration in molecules/µL (MPM) were assessed. Results KT detected 322 cases of HHV6; 10% (n=32) were HHV6A and 90% (n=290) HHV6B (Table 1). HHV6B had a higher relative abundance in children (with a distribution into adolescence) compared to adults (Figure 1). The average HHV6A MPM was 860 (range 27 - 10,472); the average HHV6B MPM was 3,361 (range 21 - 131,518). Table 1. Summary of HHV-6 Detections Figure 1. Distribution of HHV6A and HHV6B by Age Group Conclusion The distribution of the HHV6 variants detected through KT shows an overwhelming 9:1 predominance of HHV6B to HHV6A. The application of mcfDNA metagenomic sequencing for open-ended detection, variant determination and quantification of HHV6 provides more specific resolution than serological and PCR methods. KT may lend important insights into the association of specific HHV6 variants with clinical syndromes affecting vulnerable populations. Disclosures Jose Alexander, MD, D(ABMM), FCCM, CIC, SM, MB(ASCP), BCMAS, Karius (Employee) Sivan Bercovici, PhD, Karius (Employee) Nicholas R. Degner, MD, MPH, MS, Karius Inc. (Employee, Shareholder) Ricardo Castillo-Galvan, MD MPH, Karius Inc. (Consultant) Aparna Arun, MD, Karius (Employee) Ann Macintyre, DO, Karius, Inc. (Employee) Bradley Perkins, MD, Karius, Inc. (Employee) Asim A. Ahmed, MD, Karius, Inc. (Employee) Matthew Smollin, PharmD, Karius, Inc. (Employee)

Host inflammatory responses predict worse outcome in severe pneumonia, yet little is known about what drives dysregulated inflammation. We performed metagenomic sequencing of microbial cell-free DNA (mcfDNA) in 83 mechanically ventilated patients (26 culture-positive, 41 culture-negative pneumonia, 16 uninfected controls). Culture-positive patients had higher levels of mcfDNA than those with culture-negative pneumonia and uninfected controls (p

We present 10 patients with Rickettsia typhi infection in whom next-generation sequencing of microbial cell-free deoxyribonucleic acid (mcfDNA) was used as a diagnostic tool. Rickettsia typhi mcfDNA was detected in all cases and was more rapid and specific than rickettsial serology. Rickettsia typhi mcfDNA impacted antibiotic management in 50% of patients.

Background Microbial cell-free DNA (mcfDNA) sequencing of plasma can identify the presence of a pathogen in a host. In this study, we evaluated the duration of pathogen detection by mcfDNA sequencing vs conventional blood culture in patients with bacteremia. Methods Blood samples from patients with culture-confirmed bloodstream infection were collected within 24 hours of the index positive blood culture and 48 to 72 hours thereafter. mcfDNA was extracted from plasma, and next-generation sequencing was applied. Reads were aligned against a curated pathogen database. Statistical significance was defined with Bonferroni adjustment for multiple comparisons (P Results A total of 175 patients with Staphylococcus aureus bacteremia (n = 66), gram-negative bacteremia (n = 74), or noninfected controls (n = 35) were enrolled. The overall sensitivity of mcfDNA sequencing compared with index blood culture was 89.3% (125 of 140), and the specificity was 74.3%. Among patients with bacteremia, pathogen-specific mcfDNA remained detectable for significantly longer than conventional blood cultures (median 15 days vs 2 days; P Conclusions Pathogen mcfDNA identified the bacterial etiology of bloodstream infection for a significantly longer interval than conventional cultures, and its duration of detection was associated with increased risk for metastatic infection. mcfDNA could play a role in the diagnosis of partially treated endovascular infections.

We present 10 patients with Rickettsia typhi infection in whom next-generation sequencing of microbial cell-free deoxyribonucleic acid (mcfDNA) was used as a diagnostic tool. Rickettsia typhi mcfDNA was detected in all cases and was more rapid and specific than rickettsial serology. Rickettsia typhi mcfDNA impacted antibiotic management in 50% of patients.

Background Patients hospitalized with SARS-CoV2 infections (Covid-19) are frequently febrile and can become critically ill quickly often leading to intervention with antimicrobial therapy. An etiologic diagnosis of superinfections can be difficult to obtain through the usual invasive procedures because of patient instability and the desire to avoid them because they may not be tolerated by the patient. Providers may also be hesitant to embark on such interventions in order to avoid healthcare personnel (HCP) exposure to aerosols. Methods Karius Test (KT) results are presented from 30 patients who presented with Covid-19. The KT is a CLIA certified/CAP-accredited next-generation sequencing (NGS) plasma test that detects pathogen cell free DNA (cfDNA). After cfDNA is extracted and NGS performed, human reads are removed and remaining sequences are aligned to a curated database of > 1400 organisms. Organisms present above a statistical threshold are reported. Results The KT detected pathogens in the majority of patients (n=20) with COVID-19. The most common infections were herpesviruses in 60% of patients. The most common bacterial pathogen was E. coli seen in 25% of patients. 15 out of 20 patients had more than one pathogen detected. 15% of patients had fungal pathogens, including one detection of Lichtheimia ramosa, in an immunocompromised patient. The results are summarized in the table. Co-infections detected by the Karius Test in patients hospitalized with COVID-19 Conclusion Open-ended, plasma-based NGS for mcfDNA provides a non-invasive method to assess for co-infections in critically ill patients with COVID-19. This report highlights the potential to increase diagnostic yield as well as to decrease the need for invasive procedures – and their attendant risks to patients and HCP – to obtain etiologic diagnoses to better inform antimicrobial therapy for superinfection. It also serves to highlight the variety of pathogens affecting these patients during the COVID-19 pandemic. Disclosures William V. La Via, MD, Karius (Employee) Sudeb Dalai, MD, Karius (Employee) Christiaan R. de Vries, MD, PhD, Karius (Consultant, Independent Contractor)Stanford University (Employee) Ann Macintyre, DO, Karius (Employee) Asim A. Ahmed, MD, Karius (Employee)

Meningitis and encephalitis are leading causes of central nervous system (CNS) disease and often result in severe neurological compromise or death. Traditional diagnostic workflows largely rely on pathogen-specific diagnostic tests, sometimes over days to weeks. Metagenomic next-generation sequencing (mNGS) is a high-throughput platform that profiles all nucleic acid in a sample. We prospectively enrolled 68 patients from New England with known or suspected CNS infection and performed mNGS from both RNA and DNA to identify potential pathogens. Using a computational metagenomic classification pipeline based on KrakenUniq and BLAST, we detected pathogen nucleic acid in cerebrospinal fluid (CSF) from 22 subjects. This included some pathogens traditionally diagnosed by serology or not typically identified in CSF, including three transmitted by Ixodes scapularis ticks (Powassan virus, Borrelia burgdorferi, Anaplasma phagocytophilum). Among 24 subjects with no clinical diagnosis, we detected enterovirus in two subjects and Epstein Barr virus in one subject. We also evaluated two methods to enhance detection of viral nucleic acid, hybrid capture and methylated DNA depletion. Hybrid capture nearly universally increased viral read recovery. Although results for methylated DNA depletion were mixed, it allowed detection of varicella zoster virus DNA in two samples that were negative by standard mNGS. Overall, mNGS is a promising approach that can test for multiple pathogens simultaneously, with similar efficacy to pathogen-specific tests, and can uncover geographically relevant infectious CNS disease, such as tick-borne infections in New England. With further laboratory and computational enhancements, mNGS may become a mainstay of workup for encephalitis and meningitis.ImportanceMeningitis and encephalitis are leading global causes of central nervous system (CNS) disability and mortality. Current diagnostic workflows remain inefficient, requiring costly pathogen-specific assays and sometimes invasive surgical procedures. Despite intensive diagnostic efforts, 40-60% of people with meningitis or encephalitis have no clear cause of their CNS disease identified. As diagnostic uncertainty often leads to costly inappropriate therapies, the need for novel pathogen detection methods is paramount. Metagenomic next-generation sequencing (mNGS) offers the unique opportunity to circumvent these challenges using unbiased laboratory and computational methods. Here, we performed comprehensive mNGS from 68 patients with suspected CNS infection, and define enhanced methods to improve the detection of CNS pathogens, including those not traditionally identified in the CNS by nucleic acid detection. Overall, our work helps elucidate how mNGS can become a mainstay in the diagnostic toolkit for CNS infections.

Background A diverse spectrum of invasive molds and fungi cause serious opportunistic infections in immunocompromised (IC) children. Their overlap in clinical presentation can make it challenging to differentiate among etiologies and optimally tailor antifungal therapy. Current methods to identify these pathogens lack sensitivity, are limited by long turnaround times and require an array of individual tests on invasively obtained specimens. The delay or lack of a pathogen diagnosis in combination with the reliance on invasive procedures leads to a dependence on broad empiric therapy, the development of antimicrobial resistance and increases in morbidity and mortality. Rapid, non-invasive diagnosis of invasive fungal infections through next-generation sequencing (NGS) of plasma microbial cell-free DNA (mcfDNA) offers a means to overcome these limitations. Methods Karius® Test (KT) results were reviewed for detections of Aspergillus, non-Aspergillus molds and Pneumocystis jirovecii (PJP) in children. KT, developed and validated in Karius’ CLIA certified/CAP accredited lab, detects microbial cell-free DNA (mcfDNA) can assist with the diagnosis of invasive infections. McfDNA is extracted, NGS is performed, human sequences removed and remaining sequences aligned to a curated pathogen database of >1400 organisms. Organisms present above a statistical threshold are reported and quantified. For > 85% of tests the time to result reporting is 24 hours from sample receipt. Clinical information was included from data submitted with the requisition or obtained at the time of reporting from clinical consultations with the provider. Results KT detected 7 different species of Aspergillus in 61 patients (74% IC, 40% with a pulmonary focus). KT detected 15 different non-Aspergillus molds in 51 patients (80% IC, 36% with a pulmonary focus). KT detected PJP in 37 patients (73% IC, 76% with a pulmonary focus, 54% with a DNA virus co-detection and 32% with a herpesvirus co-detections). There were 31 subjects with serial monitoring (97% IC, 70% with a pulmonary focus) including 48% with Aspergillus, 39% with non-Aspergillus molds and 12% with PJP (Figure 1). 71% of subjects demonstrated a decline in the quantitative mcfDNA signal over time; the duration of a positive mcfDNA signal ranged from 3–92 days (median 16 days, SD 22.4). Conclusions Plasma mcfDNA NGS offers a rapid, non-invasive means of detecting a broad diversity of invasive pathogens that overlap in their clinical presentations and are difficult to identify in immunocompromised children. The rapid turnaround time, non-invasive sampling and 1-sample-1000+test-solution may lead to a faster time to pathogen diagnosis, faster time to targeted therapy and obviate the need for invasive diagnostic procedures. The ability with a single test to concomitantly diagnose co-pathogens including reactivating herpesviruses that modulate the progression of principal infecting fungal pathogens (i.e. cytomegalovirus modulation of PJP) can help optimize care. Additionally, this convenient non-invasive means of serial testing of invasive fungal infections may serve as an indicator of burden of infection, provide insight into treatment efficacy and ultimately help define the length and mode (medical/surgical) of therapy required to improve outcomes. Additional studies correlating the mcfDNA signal with individual patient clinical and radiographic parameters will be important to further define the utility of serial mcfDNA monitoring.

Objective: To determine the efficacy of topical treatment with 1% propranolol in the infants with hemangioma. Methodology:This was a prospective study conducted from August 2018 till December 2019 through non-probability convenient sampling technique. Study comprised of 27 infants from the Dermatology Department of Dow University Hospital, Karachi. Infants having hemangioma in any part of body except near orifical opening were included in the study. 1% topical propranolol was applied twice daily to the lesion and any change is size, colour , discharge before and after treatment was noted at each follow up till 3 months.Resolution was recorded at each follow up. Data was analysed in SPSS version 20.0. P-value ≤0.05 was taken as significant. Results:From the total of 27 infants enrolled in the study, 09 (33.3%) were male and 18 were female (66.7%). After treatment, 26 (96.3%)hemangiomas had shrunken. 14 (51.9%) of patients parents termed it as a very good response to treatment and 07 (25.9%) felt complete satisfaction to treatment. On first follow up, only 01 (3.7%) had complete resolution, 04 (14.8%) on second follow up and 06 (22.2%) on third follow up. Conclusion: The present study concluded that 1% topical propranolol was effective as well as safe and convenient to apply for treating infantile hemangiomas.&nbsp

Background Laboratory confirmation of early Lyme borreliosis (LB) is challenging. Serology is insensitive during the first days to weeks of infection, and blood polymerase chain reaction (PCR) offers similarly poor performance. Here, we demonstrate that detection of Borrelia burgdorferi (B.b.) cell-free DNA (cfDNA) in plasma can improve diagnosis of early LB. Methods B.b. detection in plasma samples using unbiased metagenomic cfDNA sequencing performed by a commercial laboratory (Karius Inc) was compared with serology and blood PCR in 40 patients with physician-diagnosed erythema migrans (EM), 28 of whom were confirmed to have LB by skin biopsy culture (n = 18), seroconversion (n = 2), or both (n = 8). B.b. sequence analysis was performed using investigational detection thresholds, different from Karius’ clinical test. Results B.b. cfDNA was detected in 18 of 28 patients (64%) with laboratory-confirmed EM. In comparison, sensitivity of acute-phase serology using modified 2-tiered testing (MTTT) was 50% (P = .45); sensitivity of blood PCR was 7% (P = .0002). Combining B.b. cfDNA detection and MTTT increased diagnostic sensitivity to 86%, significantly higher than either approach alone (P ≤ .04). B.b. cfDNA sequences matched precisely with strain-specific sequence generated from the same individual’s cultured B.b. isolate. B.b. cfDNA was not observed at any level in plasma from 684 asymptomatic ambulatory individuals. Among 3000 hospitalized patients tested as part of clinical care, B.b. cfDNA was detected in only 2 individuals, both of whom had clinical presentations consistent with LB. Conclusions This is the first report of B.b. cfDNA detection in early LB and a demonstration of potential diagnostic utility. The combination of B.b. cfDNA detection and acute-phase MTTT improves clinical sensitivity for diagnosis of early LB.

Granulomatous amoebic encephalitis (GAE) caused by Balamuthia mandrillaris is a rare subacute infection with exceptionally high mortality. Diagnosis is typically made by brain biopsy or at autopsy. Detection of Balamuthia mandrillaris cell-free DNA by next-generation sequencing of plasma enabled rapid, noninvasive diagnosis in a case of amoebic encephalitis.

Hypertension is an increasingly global issue and causes multiple complications if not controlled early. Objective: To see the prevalence of hypertension among the patients presenting in the outdoor department. Material and Methods: This cross-sectional study included 134 patients of either gender. Patient of age 18 years and above were included. Data was collected and analyzed using SPSS V. 23. Results: Mean age of the patients was 38.98±13.35 years. There were 69 female patients (51.49%) and 65 male patients (48.51%). Mean systolic blood pressure was 140.01±20.15 mmHg with a minimum value of 110 mmHg and a maximum value of 175 mmHg. Mean diastolic blood pressure was 88.21±5.45 mmHg with a minimum value of 80 mmHg and a maximum value of 99 mmHg. Nineteen (14.18%) patients were having normal systolic blood pressure, 23 (17.16%) patients were suffering from pre-hypertension, 50 (37.31%) were class I hypertensive and and 42 (31.34%) were class II hypertensive. Conclusion: This study concludes the higher frequency of hypertension in patients presenting in the outdoor department. Keywords: Hypertension, outdoor department, Pakistan. &nbsp

Background Coxiella burnetii and Brucella spp. are zoonotic bacterial pathogens responsible for Q fever and Brucellosis, respectively. Both pathogens have a global distribution and Brucellosis is the most common zoonosis in the world. However, the CDC reports only 80-120 cases of human brucellosis and ~150 cases of acute Q fever annually. The diagnosis of these infections can be limited by: (1) their difficulty to culture; (2) the insensitivity and nonspecificity of serology; (3) the clinical overlap with other infections; and (4) the unreliability of epidemiological exposure history for these zoonoses. Unbiased microbial cell free DNA (mcfDNA) next-generation sequencing (NGS) offers a potential solution to overcome these limitations. Methods The Karius TestTM (KT) developed and validated in Karius’s CLIA certified/CAP accredited lab in Redwood City, CA detects mcfDNA in plasma. After mcfDNA is extracted and NGS performed, human reads are removed, and remaining sequences are aligned to a curated database of > 1500 organisms. McfDNA from organisms present above a statistical threshold are reported and quantified in molecules/µL (MPM). KT detections of Coxiella and Brucella were reviewed from August 2017 - present; clinical information was obtained with test requisition or consultation upon result reporting. Results KT detected 8 cases of Coxiella burnetii (1735 MPM +/- 3000) and 5 cases of Brucella melitensis (avg 296 MPM +/- 223) (Table 1), representing approximately 1-2% of all detections in the US during this period. All of the Coxiella detections were in adults (100% male) with 5 cases of fever of unknown origin, 2 cases of culture-negative endocarditis and one case of endovascular graft infection. Brucella detections occurred in 3 adults and 2 children (60% male), 3 with exposure to unpasteurized dairy and included 3 cases of spine infection (2 vertebral osteomyelitis, 1 epidural abscess). Conclusion Open-ended, plasma-based mcfDNA NGS provides a rapid, non-invasive test to diagnose diverse clinical manifestations of zoonotic infections such as Q fever and Brucellosis against competing broad differential diagnoses. Furthermore, these cases highlight the potential of the KT to diagnose infections caused by fastidious/unculturable pathogens with cryptic clinical presentations. Disclosures Nicholas R. Degner, MD, MPH, MS, Karius Inc. (Employee, Shareholder) Ricardo Castillo-Galvan, MD MPH, Karius Inc. (Consultant) Jose Alexander, MD, D(ABMM), FCCM, CIC, SM, MB(ASCP), BCMAS, Karius (Employee) Aparna Arun, MD, Karius (Employee) Ann Macintyre, DO, Karius, Inc. (Employee) Bradley Perkins, MD, Karius, Inc. (Employee) Asim A. Ahmed, MD, Karius, Inc. (Employee) Matthew Smollin, PharmD, Karius, Inc. (Employee)