Georgiou, P

Herberg, J

Kaforou, M

Levin, M

Moniri, A

Pennisi, I

Rodriguez Manzano, J

JAMA Pediatrics

English

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out of the analysis. Still, the states included in our analyses aregeographically representative and include more than 29 mil-lion children in the United States. As conversations around in-person education continue, hospitalization growth may offerreasons for concern.Zachary Levin, BAKimberly Choyke, MSArchelle Georgiou, MDSoumya Sen, PhDPinar Karaca-Mandic, PhDAuthor Affiliations: Division of Health Policy and Management, University ofMinnesota School of Public Health, Minneapolis (Levin); Department ofFinance, Medical Industry Leadership Institute, University of MinnesotaCarlson School of Management, Minneapolis (Choyke, Karaca-Mandic);Starkey Hearing Technologies, Eden Prairie, Minnesota (Georgiou);Department of Information and Decision Sciences, University of MinnesotaCarlson School of Management, Minneapolis (Sen).Corresponding Author: Pinar Karaca-Mandic, PhD, Carlson School ofManagement, 321 19th Ave S, Minneapolis, MN 55455 (pkmandic@umn.edu).Accepted for Publication: September 23, 2020.Published Online: January 11, 2021.doi:10.1001/jamapediatrics.2020.5535Author Contributions: Dr Karaca-Mandic had full access to all the datain the study and takes responsibility for the integrity of the dataand the accuracy of the data analysis.Concept and design: All authors.Acquisition, analysis, or interpretation of data: All authors.Drafting of the manuscript: Levin, Choyke, Georgiou, Karaca-Mandic.Critical revision of the manuscript for important intellectual content:All authors.Statistical analysis: Levin, Choyke, Sen, Karaca-Mandic.Obtained funding: Karaca-Mandic.Administrative, technical, or material support: Choyke, Sen.Supervision: Georgiou, Karaca-Mandic.Conflict of Interest Disclosures: Dr Karaca-Mandic reports funding from theUniversity of Minnesota, Office of Academic Clinical Affairs, and grants fromUnited Health Foundation during the conduct of the study; personal fees fromTactile Medical, Precision Health Economics, and Sempre Health; and grantsfrom Agency for Healthcare Research and Quality, the American Cancer Society,the National Institute for Health Care Management, the National Institute onDrug Abuse, and the National Institutes of Health outside the submitted work.Dr Georgiou reports personal fees from HealthGrades outside the submittedwork. No other disclosures were reported.Funding/Support: This research uses publicly available data from the Universityof Minnesota COVID-19 Hospitalization Project, which is partially funded by theUniversity of Minnesota Office of Academic Clinical Affairs and United HealthFoundation. Although the age breakdown of hospitalizations are not reported onthe project website, data can be requested from the project team.Role of the Funder/Sponsor: The funding sources had no role in the design andconduct of the study; collection, management, analysis, and interpretation ofthe data; preparation, review, or approval of the manuscript; and decision tosubmit the manuscript for publication.Additional Contributions: Yi Zhu, MA, doctoral student at the Carlson Schoolof Management, University of Minnesota, contributed to this project by helpinglaunch the University of Minnesota COVID-19 Hospitalization Project website. YiZhu did not receive compensation. Khoa Vu, doctoral student in AppliedEconomics, University of Minnesota, provided research assistance with datacollection. Mr Vu was compensated as a research assistant.1. University of Minnesota COVID-19 Hospitalization Tracking Project. AccessedDecember 1, 2020. https://carlsonschool.umn.edu/mili-misrc-covid19-tracking-project2. Census Bureau. Single Year of Age and Sex Population Estimates:April 1, 2010 to July 1, 2019. Accessed December 1, 2020. https://www.census.gov/data/tables/time-series/demo/popest/2010s-state-detail.htmlTranslation of a Host Blood RNA SignatureDistinguishing Bacterial From Viral InfectionInto a Platform Suitable for Developmentas a Point-of-Care TestDiscrimination of viral from bacterial infections remains a chal-lenge, resulting in unnecessary investigation, admission, andantibiotic treatment of many patients with fever. Studies inJAMA previously reported that children with bacterial and vi-ral infection can be distinguished by their blood host RNAsignature.1,2 Here, we demonstrate that a 2-gene RNA signa-ture can be translated into a rapid (<25 minutes) and portablelaboratory-on-a-chip platform suitable for development as apoint-of-care test.Methods | Herberg and colleagues1,3 reported a 2-transcriptsignature (IFI44L and FAM89A) discovered using geneexpression microarrays in a set of 455 children with feverand bacterial and viral infections (Inflammatory and Infec-tious Disease Study [IRIS] study). We randomly selected24 RNA samples from patients in the IRIS study1 withconfirmed bacterial (n = 12) and viral (n = 12) infections,matched for severity, collected between September 2009and May 2017, and extracted using the PAXgene Blood RNAKit (PreAnalytiX GmbH). We identified transcripts suitablefor translation to our laboratory-on-chip platform, whichuses reverse transcription loop-mediated isothermal ampli-fication (RT-LAMP), by assessing counts of IFI44L andFAM89A in a publicly available blood RNA sequencing dataset comprising 255 children with bacterial and viral infec-tions (GSE69529).4 The average gene counts for IFI44L weresufficient (2281.9) but low for FAM89A (20.2), potentiallycompromising transferability across platforms. Therefore,using the list of previously identified 38 highly correlatedtranscripts,1 we replaced FAM89A with EMR1-ADGRE1(RefSeq ID: NM_001974.5), which had sufficient averagegene counts (511.3),4 and in combination with IFI44L(RefSeq ID: NM_006820) had a similar performance to theoriginal 2 transcript signature (area under the curve [AUC]in the training, test, and validation data sets: 93.4%, 97.4%,and 97.2%, respectively).Our laboratory-on-chip platform combines novelpH-sensing complementary metal-oxide semiconductortechnology5 with RT-LAMP, which we term electronicRT-LAMP (RT-eLAMP) (Figure 1). RT-eLAMP uses thousandsof microsensors (ion-sensitive field-effect transistors) todetect H+ ions released, resulting in a change in pH, duringthe nucleic acid amplification process following the sameexperimental conditions as reported in our previousarticle.6We compared the gene expression values (normalizedlog2 fluorescence for microarrays1) or time-to-positivevalues from signals of all the microsensors for RT-eLAMPusing support vector classifiers, with 10-fold cross-validation, and each patient was assigned a score re-flecting risk of bacterial or viral disease (package: scikit-learn in Python). We evaluated the predictive accuracy ofthe score in patients with microbiologically confirmed diag-noses using receiver operating characteristic curves, AUC,Lettersjamapediatrics.com (Reprinted) JAMA Pediatrics April 2021 Volume 175, Number 4 417Downloaded From: https://jamanetwork.com/ by a Imperial College London User  on 04/09/2021and 95% CIs under the binomial distribution. Analysisbegan April 2020.Results | Using microarray data,1 the 2-transcript signatureapplied to children with bacterial and viral infections had asensitivity and specificity of 100% (95% CI, 95.7%-100%)and 100% (95% CI, 95.7%-100%), respectively, with AUC of100% (95% CI, 91.5%-100%) (Figure 2A and B). Translatingthe 2-gene signature to RT-eLAMP showed similar results:sensitivity and specificity were 100% (95% CI, 96.0%-100%)and 100% (95% CI, 96.0%-100%), respectively, with AUC of100% (95% CI, 92.2%-100%) (Figure 2C and D). The calcu-lated limit of detection for the 2 assays were 10 RNA copiesper reaction in 10-μL reaction volume and time to positiveunder 25 minutes, much faster than microarray.Discussion | Previously reported RNA signatures that differ-entiate bacterial from viral infection were discovered usingcumbersome transcriptomic technologies. We have movedthe promising transcript signatures closer to clinical appli-cation by establishing that the 2-transcript signature can bedetected using a semiconductor-based sensing platformcombined with isothermal amplification chemistries. Theabsence of fluorescent labels and the economy of scale ofthe microchip industry makes the technology potentiallysuitable for implementation at low cost (<£1 [US $1.33] perchip). While our study includes modest patient numbers,it provides a proof of concept that host RNA signatures canbe detected rapidly and cost-effectively in a format suitablefor development as a point-of-care diagnostic test thatmight be applied to a range of clinical diagnoses. This studyhas a number of limitations including a relatively smallsample size, a focus on confirmed infections, and that theplatform currently lacks integrated sample preparation andhas a limited numbers of wells per microchip.Ivana Pennisi, MScJesus Rodriguez-Manzano, PhDAhmad Moniri, MEngMyrsini Kaforou, PhDJethro A. Herberg, PhDMichael Levin, PhDPantelis Georgiou, PhDAuthor Affiliations: Department of Infectious Disease, Faculty of Medicine,Imperial College London, London, United Kingdom (Pennisi,Rodriguez-Manzano, Kaforou, Herberg, Levin); Centre for Bio-InspiredTechnology, Department of Electrical and Electronic Engineering,Imperial College London, London, United Kingdom (Moniri, Georgiou).Corresponding Author: Michael Levin, PhD, Section of Paediatrics,Department of Infectious Disease, Faculty of Medicine, Imperial CollegeFigure 1. A Handheld Point-of-Care TestDisposable diagnostic cartridge containing a complementary metal-oxidesemiconductor ion-sensitive field-effect transistor array for on-chip real-timenucleic acid amplification.Figure 2. Performance of the 2 Diagnostic Genes in Microarrayand Electronic Reverse Transcription Loop-Mediated IsothermalAmplification (RT-eLAMP)420–2–4–6–8ScoreMicroarray scoreADefinitebacterial infection(n =11)Definiteviral infection(n =11)7.55.02.50–2.5–5.0–7.5–10.0–12.5ScoreRT-eLAMP scoreBDefinitebacterial infection(n =12)Definiteviral infection(n=12)Microarray ROC curveC RT-eLAMP ROC curveD1.00.80.60.40.20Sensitivity0 0.2 0.4 0.6 0.8 1.01–Specificity1.00.80.60.40.20Sensitivity0 0.2 0.4 0.6 0.8 1.01–SpecificityAUC: 100% (95% CI, 92.2%-100%)AUC: 100% (95% CI, 91.5%-100%)95% CIMean ROC95% CIMean ROCPredicitive score, receiver operating characteristic (ROC) curves, and expressionvalues based on the 2-transcript signature for distinguishing definite viral (darkblue) and definite bacterial (light blue) samples. Boxes indicate the interquartileranges and the median (middle line). Whiskers indicate 1.5 times theinterquartile range. A and C, Performance using expression values obtainedfrom the microarray platform. B and D, Performance using our RT-eLAMPplatform. AUC indicates area under the curve.Letters418 JAMA Pediatrics April 2021 Volume 175, Number 4 (Reprinted) jamapediatrics.comDownloaded From: https://jamanetwork.com/ by a Imperial College London User  on 04/09/2021London, Norfolk Place, London, W2 1PG, United Kingdom(m.levin@imperial.ac.uk).Accepted for Publication: June 23, 2020.Published Online: January 4, 2021. doi:10.1001/jamapediatrics.2020.5227Open Access: This is an open access article distributed under the termsof the CC-BY License. © 2021 Pennisi I et al. JAMA Pediatrics.Author Contributions: Drs Levin and Georgiou had full access to all ofthe data in the study and take responsibility for the integrity of the dataand the accuracy of the data analysis.Concept and design: Pennisi, Rodriguez-Manzano,Kaforou, Levin, Georgiou.Acquisition, analysis, or interpretation of data: Pennisi,Rodriguez-Manzano, Moniri, Kaforou, Herberg, Georgiou.Drafting of the manuscript: Pennisi, Rodriguez-Manzano, Moniri,Kaforou, Levin, Georgiou.Critical revision of the manuscript for important intellectual content: Pennisi,Rodriguez-Manzano, Moniri, Kaforou, Herberg, Georgiou.Statistical analysis: Moniri, Kaforou.Obtained funding: Kaforou, Levin, Georgiou.Administrative, technical, or material support: Pennisi,Rodriguez-Manzano, Herberg, Georgiou.Supervision: Rodriguez-Manzano, Kaforou, Levin, Georgiou.Conflict of Interest Disclosures: Drs Kaforou, Herberg, and Levin report apatent to a method of identifying a patient having a bacterial infectionpending. Dr Levin reports a patent for RNA signature distiguishing bacterialand viral infection pending. All the authors report a patent for amethod to classify RNA host-response signatures using microchip technologypending.Funding/Support: This work has been funded by Rosetrees Trust (grantsP78740 and P78699). Mr Moniri is funded by the Engineering and PhysicalSciences Research Council Doctoral Training Partnership (EP/N509486/1).Dr Kaforou is funded by the Wellcome Trust (Sir Henry Wellcome Fellowshipgrant 206508/Z/17/Z). Drs Kaforou, Herberg, and Levin receive supportfrom the National Institute for Health Research Imperial College BiomedicalResearch Centre (grant DMPED P26077).Role of the Funder/Sponsor: The funders had no role in the design andconduct of the study; collection, management, analysis, and interpretation ofthe data; preparation, review, or approval of the manuscript; and decisionto submit the manuscript for publication.Additional Contributions: We thank the following persons affiliated withImperial College London, Departments of Infectious Disease and Electricaland Electronic Engineering: D. Habgood-Coote, MSc, for bioinformaticsanalysis of gene expression data sets and N. Miscourides, PhD,and N. Moser, PhD, for their contributions on the on-chip RNA detection.No compensation was received.1. Herberg JA, Kaforou M, Wright VJ, et al; IRIS Consortium. Diagnostic testaccuracy of a 2-transcript host RNA signature for discriminating bacterialvs viral infection in febrile children. JAMA. 2016;316(8):835-845. doi:10.1001/jama.2016.112362. Kaforou M, Herberg JA, Wright VJ, Coin LJM, Levin M. Diagnosis ofbacterial infection using a 2-transcript host RNA signature in febrile infants 60days or younger. JAMA. 2017;317(15):1577-1578. doi:10.1001/jama.2017.13653. Herberg JA, Kaforou M, Gormley S, et al. Transcriptomic profiling inchildhood H1N1/09 influenza reveals reduced expression of proteinsynthesis genes. J Infect Dis. 2013;208(10):1664-1668. doi:10.1093/infdis/jit3484. DeBerg HA, Zaidi MB, Altman MC, et al. Shared and organism-specific hostresponses to childhood diarrheal diseases revealed by whole blood transcriptprofiling. PLoS One. 2018;13(1):e0192082. doi:10.1371/journal.pone.01920825. Toumazou C, Shepherd LM, Reed SC, et al. Simultaneous DNA amplificationand detection using a pH-sensing semiconductor system. Nat Methods.2013;10(7):641-646. doi:10.1038/nmeth.25206. Malpartida-Cardenas K, Miscourides N, Rodriguez-Manzano J, et al.Quantitative and rapid plasmodium falciparum malaria diagnosis andartemisinin-resistance detection using a CMOS lab-on-chip platform.Biosens Bioelectron. 2019;145:111678. doi:10.1016/j.bios.2019.111678Solitary Use of Alcohol and Marijuanaby US 12th Grade Students, 1976-2019Recent reviews have highlighted adolescent solitary alcoholand marijuana use as risk indicators associated with nega-tive consequences, coping motives, and negative affect1,2;solitary use may reflect self-medication.1,2 Adolescent soli-tary alcohol use is associated with health and academicproblems,3 deviant behavior,3 and alcohol use disorder.4Data on sex differences in solitary alcohol and marijuanause have been mixed.1,2 Nationally representative estimatesof prevalence and change in adolescent solitary alcohol andmarijuana use are needed.1 This study provides 2018-2019prevalence estimates of and 1976-2019 trends in solitaryalcohol and marijuana use among all 12th grade studentsand those who used alcohol and marijuana in the past 12months, separated by sex.Methods | Data were collected from spring 1976 through spring2019 through the US nationally representative Monitoring theFuture study.5 Informed consent (active or passive, per schoolpolicy) was obtained from parents (if students were <18 years)or students (if students were ≥18 years). A University of Michi-gan institutional review board approved the study. Mean stu-dent response rates were 82.4%.5 Solitary use was asked on 1of 6 randomly distributed questionnaires. Respondents self-reported past 12-month alcohol and marijuana use and howoften such use occurred when alone. For each substance, a di-chotomous measure was coded, indicating any solitary use.Prevalence and SE estimates (calculating means of 2-yeargroupings to enhance stability) were obtained using survey pro-cedures in SAS 9.4 (SAS Institute Inc), accounting for the com-plex sample design. Using obtained estimates, models6 esti-mated linear change over time using Joinpoint version 4.7.0.0(National Cancer Institute); statistical significance was de-fined as P < .05 (2 sided).Results | Of the 121 279 students responding to the relevantquestionnaire, 9171 (7.6%) had missing data on sex; another1482 (1.2%) had missing data on past-12-month alcoholand/or marijuana use or solitary use, leaving 110 626responses. The sample included 57 257 female students(51.8%). Among all 12th grade students in 2018-2019, 14.8%(95% CI, 13.4%-16.3%) reported solitary alcohol use and15.8% (95% CI, 14.2%-17.4%) reported solitary marijuanause. Among those who had used alcohol in the past 12months in 2018-2019, solitary drinking was reported by23.5% (95% CI, 20.4%-26.6%) of female students and 30.0%(95% CI, 26.1%-33.9%) of male students; percentages forsolitary marijuana use among those who used marijuanawere 42.3% (95% CI, 37.7%-47.0%) of female students and54.8% (95% CI, 50.0%-59.6%) of male students.Trend analyses (Figure; Table) showed among all stu-dents, solitary alcohol use decreased nonsignificantly from1976-1977 through 1986-1987 (slope 1 [SE], −0.547 [0.254];P = .05), showed a slope not significantly different than 0through 1992-1993, and then decreased significantlythrough 2018-2019 (slope 3 [SE], −0.182 [0.060]; P = .009).Solitary marijuana use among all students decreased signifi-Lettersjamapediatrics.com (Reprinted) JAMA Pediatrics April 2021 Volume 175, Number 4 419© 2020 American Medical Association. 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Translation of a host blood RNA Signature distinguishing bacterial from viral infection into a platform suitable for development as a point-of-care test

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Translation of a host blood RNA Signature distinguishing bacterial from viral infection into a platform suitable for development as a point-of-care test

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