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Background Shotgun sequencing-based metagenomics is a useful approach to profiling microbiomes in environmental and patient samples. In a clinical setting, metagenomic techniques have the advantage of identifying organisms, which cannot be readily cultured or confirmed by other techniques. We have developed a clinical-grade, streamlined metagenomics-based pipeline that includes regulatory compliant method considerations, such as an internal control followed by a machine-based learning (ML) process to identify pathogens in urine samples. Methods We built an optimized novel end-to-end NGS assay pipeline that harnesses pathogen-specific genome data to detect bacterial species. We processed de-identified clinical urine specimens, collected from patients symptomatic for urinary tract infection (UTI). This workflow includes an IPC, QIACube-MDx extraction, library preparation and Illumina NextSeq 550 sequencing and a novel interpretable ML based analytic approach, Biotia-DX. Clinical culture results and qPCR were used as a baseline for the assay to train the ML model and to establish accuracy relative to the clinical standard of care. Results We clinically validated over 40 key uropathogens and conducted clinical studies of specificity, intra/inter reproducibility, accuracy in urine specimens (n=300), and limit of detection in E. coli, K. pneumoniae, P. mirabilis, S. aureus, E. faecalis and Candida. Additionally, the implementation of an internal control coupled with our Biotia-DX software provides an accurate (F1 score 94.3%) and highly sensitive clinical grade diagnostic tool. Conclusion Urine has historically presented a challenge for diagnostics via culturing, with a high rate of culture-negative results (∼30% on average). We improved the clinical utility of an NGS urine assay by leveraging an IPC and ML software. This decreased the rate of false positive species called in a sample relative to other NGS techniques and allows for greater sensitivity and taxonomic specificity. This assay may be especially useful for low colony-count or negative-culture samples to diagnose and guide patient treatment. Disclosures Mara Couto-Rodriguez, MS, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company David C. Danko, PhD, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company Xavier O. Jirau Serrano, MS, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company Taylor Paisie, MS, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company John C. Papciak, BS, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company Eszter Szollosi, BS, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company Christopher E. Mason, PhD, Biotia Inc.: Advisor/Consultant|Biotia Inc.: Board Member|Biotia Inc.: Ownership Interest Caitlin Otto, PhD, D(ABMM), Biotia Inc.: Advisor/Consultant Niamh B. O'Hara, PhD, Biotia Inc.: Board Member|Biotia Inc.: Ownership Interest Dorottya Nagy-Szakal, MD PhD, Biotia Inc.: Employee of Biotia Inc. a for-profit biotechnology company|Biotia Inc.: Stocks/Bonds.

The emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genetic variants that may alter viral fitness highlights the urgency of widespread next-generation sequencing (NGS) surveillance. To profile genetic variants of the entire SARS-CoV-2 genome, we developed and clinically validated a hybridization capture SARS-CoV-2 NGS assay, integrating novel methods for panel design using double-stranded DNA (dsDNA) biotin-labeled probes, and built accompanying software. This test is the first hybrid capture-based NGS assay given Food and Drug Administration (FDA) emergency use authorization for detection of the SARS-CoV-2 virus. The positive and negative percent agreement (PPA and NPA, respectively) were defined in comparison to the results for an orthogonal real-time reverse transcription polymerase chain reaction (RT-PCR) assay (PPA and NPA, 96.7 and 100%, respectively). The limit of detection was established to be 800 copies/ml with an average fold enrichment of 46,791. Furthermore, utilizing the research-use-only analysis to profile the variants, we identified 55 novel mutations, including 11 in the functionally important spike protein. Finally, we profiled the full nasopharyngeal microbiome using metagenomics and found overrepresentation of 7 taxa and evidence of macrolide resistance in SARS-CoV-2-positive patients. This hybrid capture NGS assay, coupled with optimized software, is a powerful approach to detect and comprehensively map SARS-CoV-2 genetic variants for tracking viral evolution and guiding vaccine updates. IMPORTANCE This is the first FDA emergency-use-authorized hybridization capture-based next-generation sequencing (NGS) assay to detect the SARS-CoV-2 genome. Viral metagenomics and the novel hybrid capture NGS-based assay, along with its research-use-only analysis, can provide important genetic insights into SARS-CoV-2 and other emerging pathogens and improve surveillance and early detection, potentially preventing or mitigating new outbreaks. Better understanding of the continuously evolving SARS-CoV-2 viral genome and the impact of genetic variants may provide individual risk stratification, precision therapeutic options, improved molecular diagnostics, and population-based therapeutic solutions.

In less than nine months, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) killed over a million people, including >25,000 in New York City (NYC) alone. The COVID-19 pandemic caused by SARS-CoV-2 highlights clinical needs to detect infection, track strain evolution, and identify biomarkers of disease course. To address these challenges, we designed a fast (30-minute) colorimetric test (LAMP) for SARS-CoV-2 infection from naso/oropharyngeal swabs and a large-scale shotgun metatranscriptomics platform (total-RNA-seq) for host, viral, and microbial profiling. We applied these methods to clinical specimens gathered from 669 patients in New York City during the first two months of the outbreak, yielding a broad molecular portrait of the emerging COVID-19 disease. We find significant enrichment of a NYC-distinctive clade of the virus (20C), as well as host responses in interferon, ACE, hematological, and olfaction pathways. In addition, we use 50,821 patient records to find that renin–angiotensin–aldosterone system inhibitors have a protective effect for severe COVID-19 outcomes, unlike similar drugs. Finally, spatial transcriptomic data from COVID-19 patient autopsy tissues reveal distinct ACE2 expression loci, with macrophage and neutrophil infiltration in the lungs. These findings can inform public health and may help develop and drive SARS-CoV-2 diagnostic, prevention, and treatment strategies.

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus responsible for coronavirus disease 2019 [COVID-19]) pandemic has led to a global slowdown with far-reaching financial and social impacts. The SARS-CoV-2 respiratory virus is primarily transmitted from person to person through inhalation of infected droplets or aerosols. However, some studies have shown that virions can remain infectious on surfaces for days and can lead to human infection from contact with infected surfaces. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end study showed that the effective combination for monitoring SARS-CoV-2 on surfaces required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from surfaces. This comprehensive study can provide valuable information regarding surface monitoring of various materials as well as the capacity to retain viral RNA and allow for effective disinfection., Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is a respiratory virus primarily transmitted person to person through inhalation of droplets or aerosols, laden with viral particles. However, as recent studies have shown, virions can remain infectious for up to 72 h on surfaces, which can lead to transmission through contact. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end (E2E) study showed that the effective combination for monitoring SARS-CoV-2 on surfaces includes using an Isohelix swab collection tool, DNA/RNA Shield as a preservative, an automated system for RNA extraction, and reverse transcriptase quantitative PCR (RT-qPCR) as the detection assay. Using this E2E approach, this study showed that, in some cases, noninfectious viral fragments of SARS-CoV-2 persisted on surfaces for as long as 8 days even after bleach treatment. Additionally, debris associated with specific built environment surfaces appeared to inhibit and negatively impact the recovery of RNA; Amerstat demonstrated the highest inhibition (>90%) when challenged with an inactivated viral control. Overall, it was determined that this E2E protocol required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from test surfaces. Despite our findings of viral fragment longevity on surfaces, when this method was employed to evaluate 368 samples collected from various built environmental surfaces, all samples tested negative, indicating that the surfaces were either void of virus or below the detection limit of the assay. IMPORTANCE The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (the virus responsible for coronavirus disease 2019 [COVID-19]) pandemic has led to a global slowdown with far-reaching financial and social impacts. The SARS-CoV-2 respiratory virus is primarily transmitted from person to person through inhalation of infected droplets or aerosols. However, some studies have shown that virions can remain infectious on surfaces for days and can lead to human infection from contact with infected surfaces. Thus, a comprehensive study was conducted to determine the efficiency of protocols to recover SARS-CoV-2 from surfaces in built environments. This end-to-end study showed that the effective combination for monitoring SARS-CoV-2 on surfaces required a minimum of 1,000 viral particles per 25 cm2 to successfully detect virus from surfaces. This comprehensive study can provide valuable information regarding surface monitoring of various materials as well as the capacity to retain viral RNA and allow for effective disinfection.

Postinfectious hydrocephalus (PIH), which often follows neonatal sepsis, is the most common cause of pediatric hydrocephalus worldwide, yet the microbial pathogens underlying this disease remain to be elucidated. Characterization of the microbial agents causing PIH would enable a shift from surgical palliation of cerebrospinal fluid (CSF) accumulation to prevention of the disease. Here, we examined blood and CSF samples collected from 100 consecutive infant cases of PIH and control cases comprising infants with non-postinfectious hydrocephalus in Uganda. Genomic sequencing of samples was undertaken to test for bacterial, fungal, and parasitic DNA; DNA and RNA sequencing was used to identify viruses; and bacterial culture recovery was used to identify potential causative organisms. We found that infection with the bacterium Paenibacillus, together with frequent cytomegalovirus (CMV) coinfection, was associated with PIH in our infant cohort. Assembly of the genome of a facultative anaerobic bacterial isolate recovered from cultures of CSF samples from PIH cases identified a strain of Paenibacillus thiaminolyticus. This strain, designated Mbale, was lethal when injected into mice in contrast to the benign reference Paenibacillus strain. These findings show that an unbiased pan-microbial approach enabled characterization of Paenibacillus in CSF samples from PIH cases, and point toward a pathway of more optimal treatment and prevention for PIH and other proximate neonatal infections.

OBJECTIVES: To evaluate the association of pre-pregnancy body mass index (BMI) and gestational weight gain (GWG) on infant gut microbiota diversity and abundance and the role of breastfeeding on this association. METHODS: Prospective cohort conducted in Rio de Janeiro, Brazil. Maternal pre-pregnancy BMI (< 25; ≥ 25 kg/m², normal/excessive) and GWG adequacy (adequate; excessive) were the exposures and breastfeeding practice status (exclusively breastfeeding EBF; predominant/complementary feeding PCF) was analyzed as an effect modifier. Infant stool samples were collected for 56 infants between 26–45 days. Samples were sequenced using 16S rRNA gene sequencing (MiSeq). Analysis included alpha diversity indexes (Shannon, Faith-PD, and Observed species), beta diversity metrics and Wilcoxon-Mann-Whitney Test, linear regression, permutational multivariate analysis of variance and linear discriminant analysis effect size. RESULTS: A higher median alpha diversity in infants born from mothers with excessive GWG was observed (Mann-Whitney Test: P = 0.005) and infants born from mothers with excessive GWG were positively associated with alpha diversity (β = 0.351; SE = 0.146; p-value = 0.020). Gut microbiota of infants born from mothers with excessive pre-pregnancy BMI were enriched with Dialister genus and Lactobacillus Ruminis, Haemophilus Parainfluenzae and Veillonella Parvula species and those born from mother with excessive GWG had higher abundance of Staphylocococcus genus, Staphylococcaceae family, Bacillales order and Bacilli class. Infant gut microbiota diversity and abundance did not differ according to combined categories of pre-pregnancy BMI and breastfeeding status and GWG and breastfeeding. CONCLUSIONS: Maternal gestational weight gain was associated with diversity of the infant gut microbiota. Breastfeeding did not an effect modifier in this association. FUNDING SOURCES: Foundation for the Support of Research of the State of Rio de Janeiro, National Council for Scientific and Technological Development and Columbia University Grant.

Background COVID-19 had spread quickly, causing an international public health emergency with an alarming global shortage of COVID-19 diagnostic tests. We developed and clinically validated a next-generation sequencing (NGS)-based target enrichment assay with the COVID-DX Software tailored for the detection, characterization, and surveillance of the SARS-CoV-2 viral genome. Methods The SARS-CoV-2 NGS assay consists of components including library preparation, target enrichment, sequencing, and a COVID-DX Software analysis tool. The NGS library preparation starts with extracted RNA from nasopharyngeal (NP) swabs followed by cDNA synthesis and conversion to Illumina TruSeq-compatible libraries using the Twist Library Preparation Kit via Enzymatic Fragmentation and Unique Dual Indices (UDI). The library is then enriched for SARS-CoV-2 sequences using a panel of dsDNA biotin-labeled probes, specifically designed to target the SARS-CoV-2 genome, then sequenced on an Illumina NextSeq 550 platform. The COVID-DX Software analyzes sequence results and provides a clinically oriented report, including the presence/absence of SARS-CoV-2 for diagnostic use. An additional research use only report describes the assay performance, estimated viral titer, coverage across the viral genome, genetic variants, and phylogenetic analysis. Results The SARS-CoV-2 NGS Assay was validated on 30 positive and 30 negative clinical samples. To measure the sensitivity and specificity of the assay, the positive and negative percent agreement (PPA, NPA) was defined in comparison to an orthogonal EUA RT-PCR assay (PPA [95% CI]: 96.77% [90.56%-100%] and NPA [95% CI]: 100% [100%-100%]). Data reported using our assay defined the limit of detection to be 40 copies/ml using heat-inactivated SARS-CoV-2 viral genome in clinical matrices. In-silico analysis provided >99.9% coverage across the SARS-CoV-2 viral genome and no cross-reactivity with evolutionarily similar respiratory pathogens. Conclusion The SARS-CoV-2 NGS Assay powered by the COVID-DX Software can be used to detect the SARS-CoV-2 virus and provide additional insight into viral titer and genetic variants to track transmission, stratify risk, predict outcome and therapeutic response, and control the spread of infectious disease. Disclosures Dorottya Nagy-Szakal, MD PhD, Biotia (Employee) Mara Couto-Rodriguez, MS, Biotia (Employee) Joseph Barrows, MS, Biotia, Inc. (Employee, Shareholder) Heather L. Wells, MPH, Biotia (Consultant) Marilyne Debieu, PhD, Biotia (Employee) Courteny Hager, BS, Biotia (Employee) Kristin Butcher, MS, Twist Bioscience (Employee) Siyuan Chen, PhD, Twist Bioscience (Employee) Christopher Mason, PhD, Biotia (Board Member, Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder)Twist (Other Financial or Material Support, I am CEO of Biotia and Biotia has business partnership with Twist)

BACKGROUND: Women in Africa especially young women have very high human immunodeficiency virus (HIV) incidence rates that cannot be fully explained by behavioral risks. We investigated whether genital inflammation influenced HIV acquisition in this group. METHODS: Twelve selected cytokines including 9 inflammatory cytokines and chemokines (interleukin [IL]-1alpha IL-1beta IL-6 tumor necrosis factor-alpha IL-8 interferon-gamma inducible protein-10 [IP-10] monocyte chemoattractant protein-1 macrophage inflammatory protein [MIP]-1alpha MIP-1beta) hematopoietic IL-7 and granulocyte macrophage colony-stimulating factor and regulatory IL-10 were measured prior to HIV infection in cervicovaginal lavages from 58 HIV seroconverters and 58 matched uninfected controls and in plasma from a subset of 107 of these women from the Centre for the AIDS Programme of Research in South Africa 004 tenofovir gel trial. RESULTS: HIV seroconversion was associated with raised genital inflammatory cytokines (including chemokines MIP-1alpha MIP-1beta and IP-10). The risk of HIV acquisition was significantly higher in women with evidence of genital inflammation defined by at least 5 of 9 inflammatory cytokines being raised (odds ratio 3.2; 95% confidence interval 1.3-7.9; P = .014). Genital cytokine concentrations were persistently raised (for about 1 year before infection) with no readily identifiable cause despite extensive investigation of several potential factors including sexually transmitted infections and systemic cytokines. CONCLUSIONS: Elevated genital concentrations of HIV target cell-recruiting chemokines and a genital inflammatory profile contributes to the high risk of HIV acquisition in these African women. (c) The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions please e-mail: journals.permissions@oup.com.

Evidence indicates that the densely cultivated region of northeastern China acts as a source for the wind-borne agent of Kawasaki disease (KD). KD is an acute, coronary artery vasculitis of young children, and still a medical mystery after more than 40 y. We used residence times from simulations with the flexible particle dispersion model to pinpoint the source region for KD. Simulations were generated from locations spanning Japan from days with either high or low KD incidence. The postepidemic interval (1987-2010) and the extreme epidemics (1979, 1982, and 1986) pointed to the same source region. Results suggest a very short incubation period (

Septins are important components of the cytoskeleton that are highly conserved in eukaryotes and play major roles in cytokinesis, patterning, and many developmental processes. Septins form heteropolymers which assemble into higher-order structures including rings, filaments, and gauzes. In contrast to actin filaments and microtubules, the molecular mechanism by which septins assemble is not well-understood. Here, we report that in the filamentous fungus Aspergillus nidulans, four core septins form heteropolymeric complexes. AspE, a fifth septin lacking in unicellular yeasts, interacts with only one of the core septins, and only during multicellular growth. AspE is required for proper localization of three of the core septins, and requires this same subset of core septins for its own unique cortical localization. The ΔaspE mutant lacks developmentally-specific septin higher-order structures and shows reduced spore production and slow growth with low temperatures and osmotic stress. Our results show that at least two distinct septin heteropolymer populations co-exist in A. nidulans, and that while AspE is not a subunit of either heteropolymer, it is required for assembly of septin higher-order structures found in multicellular development.