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Background: Early and accurate diagnosis of acute infection has important consequences for antibiotic stewardship, resource allocation, and clinical outcomes. However, limited sensitivity and specificity of current diagnostics lead to the erroneous prescription of antibiotics. Host-response-based diagnostics have the potential to address these challenges, but accuracy varies widely across heterogeneous global patient populations. Methods: We performed a multi-cohort analysis of 4,200 unique samples across 69 retrospective blood transcriptome datasets from 20 countries. These samples were collected from patients with acute bacterial or viral infections representing a broad spectrum of biological (age, sex, race, ethnicity, pathogen, host genetics), clinical (severity, day of presentation), and technical (Affymetrix, Agilent, Illumina) heterogeneity. We also enrolled patients with infectious diseases in two cohorts from Laos and Nepal. Findings: Current host-response-based gene signatures distinguished intracellular bacterial infection from viral infections with substantially lower accuracy. Using 69 datasets, divided into training and validation datasets, we identified an 8-gene signature that distinguished intracellular or extracellular bacterial infections from viral infections with an area under the receiver operating characteristic curve (AUROC) >0.91 (85.9% specificity, 90.2% sensitivity). In two prospective cohorts from Nepal and Laos, profiled using Fluidigm RT-PCR, the 8-gene classifier distinguished bacterial infections from viral infections with an AUROC of 0.94 (87.9% specificity, 91% sensitivity). Interpretation: The 8-gene signature met the target product profile (90% sensitivity, 80% specificity) proposed by the WHO and others for distinguishing bacterial and viral infections. The 8-gene signature should be considered for further validation and implementation. Funding Information: PK is funded in part by the Bill and Melinda Gates Foundation (OPP1113682); the National Institute of Allergy and Infectious Diseases (NIAID) grants 1U19AI109662, U19AI057229, and 5R01AI125197; Department of Defense contracts W81XWH-18-1-0253 and W81XWH1910235; and the Ralph & Marian Falk Medical Research Trust. DAR is supported by NIH/NIAID U19 AI109761 and the Thomas C. and Joan M. Merigan Endowment at Stanford University. LOMWRU is funded by the Wellcome Trust of Great Britain. AMR is funded by the National Science Foundation Graduate Research Fellowship and the Stanford Graduate Fellowship. This research was funded in whole, or in part, by the Wellcome Trust [Grant number 220211]. Declaration of Interests: AMR, SJP, TES, DAR, and PK are named as inventors on a patent application describing the 8-gene set, which has been licensed to Inflammatix. TES and SAT are employees of Inflammatix, and TES and PK are shareholders in Inflammatix. Inflammatix had no role in the design, funding, or reporting of this work. SD is currently employed by FIND. The other authors declare no conflicts of interest. Ethics Approval Statement: Ethical clearance was granted by the former Faculty of Medical Sciences Ethical Review Committee (now University of Health Sciences Ethics Committee), National University of Laos, the Oxford University Tropical Ethics Committee, and the Stanford University Institutional Review Board.

International audience; There are many outstanding questions about how to control the global COVID-19 pandemic. The information void has been especially stark in the World Health Organization Africa Region, which has low per capita reported cases, low testing rates, low access to therapeutic drugs, and has the longest wait for vaccines. As with all disease, the central challenge in responding to COVID-19 is that it requires integrating complex health systems that incorporate prevention, testing, front line health care, and reliable data to inform policies and their implementation within a relevant timeframe. It requires that the population can rely on the health system, and decision-makers can rely on the data. To understand the process and challenges of such an integrated response in an under-resourced rural African setting, we present the COVID-19 strategy in Ifanadiana District, where a partnership between Malagasy Ministry of Public Health (MoPH) and non-governmental organizations integrates prevention, diagnosis, surveillance, and treatment, in the context of a model health system. These efforts touch every level of the health system in the district—community, primary care centers, hospital—including the establishment of the only RT-PCR lab for SARS-CoV-2 testing outside of the capital. Starting in March of 2021, a second wave of COVID-19 occurred in Madagascar, but there remain fewer cases in Ifanadiana than for many other diseases (e.g., malaria). At the Ifanadiana District Hospital, there have been two deaths that are officially attributed to COVID-19. Here, we describe the main components and challenges of this integrated response, the broad epidemiological contours of the epidemic, and how complex data sources can be developed to address many questions of COVID-19 science. Because of data limitations, it still remains unclear how this epidemic will affect rural areas of Madagascar and other developing countries where health system utilization is relatively low and there is limited capacity to diagnose and treat COVID-19 patients. Widespread population based seroprevalence studies are being implemented in Ifanadiana to inform the COVID-19 response strategy as health systems must simultaneously manage perennial and endemic disease threats.

Responses by peripheral blood leukocytes may contribute to the pathogenesis of dengue hemorrhagic fever (DHF). We used DNA microarrays to reveal transcriptional patterns in the blood of 14 adults with DHF. Acute DHF was defined by an abundance of transcripts from cell cycle— and endoplasmic reticulum (ER)—related genes, suggesting a proliferative response accompanied by ER stress. Transcript-abundance levels for immunoresponse-associated genes, including cell surface markers, immunoglobulin, and innate response elements, were also elevated. Twenty-four genes were identified for which transcript abundance distinguished patients with dengue shock syndrome (DSS) from those without DSS. All the gene transcripts associated with DSS, many of which are induced by type I interferons, were less abundant in patients with DSS than in those without DSS. To our knowledge, these data provide the first snapshot of gene-expression patterns in peripheral blood during acute dengue and suggest that DSS is associated with attenuation of selected aspects of the innate host response.

Background: Several promising live attenuated virus (LAV) dengue vaccines are in development, but information about innate immune responses and early correlates of protection are lacking. Methods: We characterized human genome-wide transcripts in whole blood from 10 volunteers at 11 time-points after immunization with the dengue virus type 3 (DENV-3) component of the NIH dengue vaccine candidate TV003 and from 30 hospitalized children with acute primary DENV-3 infection. We compared day-specific gene expression patterns with subsequent neutralizing antibody (NAb) titers. Results: The transcriptional response to vaccination was largely confined to days 5-20 and was dominated by an interferon-associated signature and a cell cycle signature that peaked on days 8 and 14, respectively. Changes in transcript abundance were much greater in magnitude and scope in symptomatic natural infection than following vaccination (maximum fold-change >200 versus 21 post-vaccination; 3,210 versus 286 transcripts with significant fold-change), but shared gene modules were induced in the same sequence. The abundance of 131 transcripts on days 8 and 9 post-vaccination was strongly correlated with NAb titers measured 6 weeks post-vaccination. Conclusions: LAV dengue vaccination elicits early transcriptional responses that mirror those found in symptomatic natural infection and provide candidate early markers of protection against DENV infection.

BACKGROUND: Several promising live attenuated dengue vaccines are in development, but information about innate immune responses and early correlates of protection is lacking. METHODS: We characterized human genome-wide transcripts in whole blood from 10 volunteers at 11 time points after immunization with the dengue virus type 3 (DENV-3) component of the National Institutes of Health dengue vaccine candidate TV003 and from 30 hospitalized children with acute primary DENV-3 infection. We compared day-specific gene expression patterns with subsequent neutralizing antibody (NAb) titers. RESULTS: The transcriptional response to vaccination was largely confined to days 5–20 and was dominated by an interferon-associated signature and a cell cycle signature that peaked on days 8 and 14, respectively. Changes in transcript abundance were much greater in magnitude and scope in symptomatic natural infection than following vaccination (maximum fold-change >200 vs 21 postvaccination; 3210 vs 286 transcripts with significant fold-change), but shared gene modules were induced in the same sequence. The abundances of 131 transcripts on days 8 and 9 postvaccination were strongly correlated with NAb titers measured 6 weeks postvaccination. CONCLUSIONS: Live attenuated dengue vaccination elicits early transcriptional responses that mirror those found in symptomatic natural infection and provide candidate early markers of protection against DENV infection. CLINICAL TRIALS REGISTRATION: NCT00831012.

[This corrects the article DOI: 10.1371/journal.ppat.1005943.].

Leptospirosis causes significant morbidity and mortality worldwide; however, the role of the host immune response in disease progression and high case fatality (>10–50%) is poorly understood. We conducted a multi-parameter investigation of patients with acute leptospirosis to identify mechanisms associated with case fatality. Whole blood transcriptional profiling of 16 hospitalized Brazilian patients with acute leptospirosis (13 survivors, 3 deceased) revealed fatal cases had lower expression of the antimicrobial peptide, cathelicidin, and chemokines, but more abundant pro-inflammatory cytokine receptors. In contrast, survivors generated strong adaptive immune signatures, including transcripts relevant to antigen presentation and immunoglobulin production. In an independent cohort (23 survivors, 22 deceased), fatal cases had higher bacterial loads (P = 0.0004) and lower anti-Leptospira antibody titers (P = 0.02) at the time of hospitalization, independent of the duration of illness. Low serum cathelicidin and RANTES levels during acute illness were independent risk factors for higher bacterial loads (P = 0.005) and death (P = 0.04), respectively. To investigate the mechanism of cathelicidin in patients surviving acute disease, we administered LL-37, the active peptide of cathelicidin, in a hamster model of lethal leptospirosis and found it significantly decreased bacterial loads and increased survival. Our findings indicate that the host immune response plays a central role in severe leptospirosis disease progression. While drawn from a limited study size, significant conclusions include that poor clinical outcomes are associated with high systemic bacterial loads, and a decreased antibody response. Furthermore, our data identified a key role for the antimicrobial peptide, cathelicidin, in mounting an effective bactericidal response against the pathogen, which represents a valuable new therapeutic approach for leptospirosis., Author Summary Leptospirosis causes over one million cases and nearly 60,000 deaths annually. Infection with the spirochetal bacterium results in a spectrum of symptoms, ranging from mild febrile illness to life-threatening pulmonary hemorrhage syndrome and acute kidney injury. Despite leptospirosis being a leading cause of zoonotic morbidity worldwide, little is known about the human immune response to Leptospira infections, and less about the pathogenic mechanisms resulting in severe disease outcomes. Here, we used a systems biology approach to discover transcripts and immunoprofiles associated with case fatality. We identified new risk factors for high bacterial loads and fatal leptospirosis, including the antimicrobial peptide, cathelicidin, which we validated in an animal model. Cathelicidin therefore represents a potential novel treatment for severe cases of leptospirosis.

Background The development of a vaccine against dengue faces unique challenges, including the complexity of the immune responses to the four antigenically distinct serotypes. Genome-wide transcriptional profiling provides insight into the pathways and molecular features that underlie responses to immune system stimulation, and may facilitate predictions of immune protection. Methodology/Principal Findings In this study, we measured early transcriptional responses in the peripheral blood of cynomolgus macaques following vaccination with a live, attenuated tetravalent dengue vaccine candidate, TDV, which is based on a DENV-2 backbone. Different doses and routes of vaccine administration were used, and viral load and neutralizing antibody titers were measured at different time-points following vaccination. All 30 vaccinated animals developed a neutralizing antibody response to each of the four dengue serotypes, and only 3 of these animals had detectable serum viral RNA after challenge with wild-type dengue virus (DENV), suggesting protection of vaccinated animals to DENV infection. The vaccine induced statistically significant changes in 595 gene transcripts on days 1, 3, 5 and 7 as compared with baseline and placebo-treated animals. Genes involved in the type I interferon (IFN) response, including IFI44, DDX58, MX1 and OASL, exhibited the highest fold-change in transcript abundance, and this response was strongest following double dose and subcutaneous (versus intradermal) vaccine administration. In addition, modules of genes involved in antigen presentation, dendritic cell activation, and T cell activation and signaling were enriched following vaccination. Increased abundance of gene transcripts related to T cell activation on day 5, and the type I IFN response on day 7, were significantly correlated with the development of high neutralizing antibody titers on day 30. Conclusions/Significance These results suggest that early transcriptional responses may be predictive of development of adaptive immunity to TDV vaccination in cynomolgus macaques, and will inform studies of human responses to dengue vaccines., Author Summary Dengue has become the leading cause of mosquito-borne virus infections worldwide. Despite considerable effort, development of a successful vaccine against dengue virus (DENV) has been challenging due to the co-circulation of the four DENV serotypes in endemic areas—to which humans develop distinct immune responses, and the increased risk of severe disease in those with pre-existing immunity to one serotype when they are infected with a different serotype. In this study, we investigated the responses in macaques to vaccination with the tetravalent, live-attenuated vaccine, TDV, by different doses and routes of vaccine administration. We identify changes in macaque gene expression that occurred in the days immediately following vaccination with TDV, a time-period that is difficult to study during natural infection. The gene expression response was characterized by features of the innate immune response to virus, notably the type I interferon response, and began the day after TDV vaccination. This response correlated with the development of neutralizing antibodies, which means that it might serve as an early indicator of a subsequent protective immune response to dengue vaccines.

Background To guide treatment of infectious diseases, clinicians need sensitive, specific, and rapid diagnostics. We aim to incorporate complementary methods of microbial sequencing and host-response profiling to improve the diagnosis of patients at risk for acute infections. Methods We enrolled 200 adult patients with systemic inflammatory response syndrome (SIRS) at the Stanford Emergency Department. Physicians with specialty training in infectious diseases conducted retrospective two-physician chart review to establish likely admission diagnoses. Blood samples were tested with a previously described 18-gene host-response integrated antibiotics decision model (IADM) that distinguishes noninfectious SIRS, bacterial infections and viral infections. Plasma samples were tested with shotgun metagenomic next-generation sequencing (NGS) and viral sequencing with VirCapSeq. A novel statistical algorithm was developed to identify contaminant organism sequences in NGS data. Results The physician chart review classified 99 patients (49%) as infected, 69 (35%) possibly infected and 32 (16%) non-infected. Compared with chart review, the IADM distinguished bacterial from viral infections with an area under curve of 0.85 (95% confidence interval 0.77–0.93). NGS results to date confirmed positive blood cultures in seven of nine patients, with two of four blood culture-positive E. coli patients turning up negative on NGS due to E. coli contamination. NGS also confirmed positive cultures from other sites in two of six patients with negative blood cultures. Preliminary VirCapSeq data from 23 patients confirmed positive viral tests in five of six patients with Hepatitis C, BK Virus, Cytomegalovirus and Epstein–Barr Virus infections. VirCapSeq did not identify a causative agent in the plasma of 11 patients with confirmed respiratory viral infection and intestinal Norovirus infection, and six patients with idiopathic illness. Interestingly, VirCapSeq found viral reactivation in 8 of 12 immunocompromised patients. Conclusion The diagnosis of suspected infections may be enhanced by integrating host-response and microbial data alongside clinical judgment. Our results and large cohort lay the foundation to demonstrate the utility of this approach and in which patients these tools may be most useful. Disclosures T. E. Sweeney, Inflammatix, Inc: Employee and Shareholder, Salary; T. Briese, Roche: Columbia University has licensed VirCapSeq to Roche, Licensing agreement or royalty; W. I. Lipkin, Roche: Columbia University has licensed VirCapSeq to Roche., Licensing agreement or royalty; P. Khatri, Inflammatix, Inc.: Co-founder, Scientific Advisor and Shareholder, Licensing agreement or royalty and ownership stock; D. A. Relman, Karius: Consultant, Stock options; Arc Bio LLC: Consultant, Stock options