Search

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.

Until recently, whole genome sequencing (WGS) has been the preserve of high-income settings. Although the costs of WGS have dramatically decreased over the past decade,1 the need for initial investment in sequencing platforms and associated equipment and for the technical expertise to run and maintain them have prevented their introduction into lower income settings. Downstream processing of sequencing data is frequently hampered by poor Internet connectivity, a lack of local expertise, and the perceived requirement for substantial computational infrastructure. Oxford Nanopore Technologies’ (ONT) MinION sequencer is a portable device for DNA and RNA sequencing that generates data for local analysis, in real time. The MinION weighs under 100 g and plugs directly into a laptop via a USB port, with no additional computing infrastructure required.2 Only basic laboratory facilities are needed to extract DNA and prepare sequencing libraries. The MinION has been used to sequence viral,3 bacterial,4,5 and eukaryotic6–9 genomes. Despite its high raw error rate, nanopore data can in many cases produce highly contiguous assemblies. Illumina short read data can be added to improve consensus accuracy, in many cases to that of a high-quality draft assembly, without extra finishing steps. The Lao People’s Democratic Republic (Laos) is a land-linked country of ∼7 million people in Southeast Asia. Lao People’s Democratic Republic is ranked 135th in the world on the Human Development Index, a composite statistic of life expectancy, education, and per capita income.10 There are few functioning molecular biology laboratories in Laos, no previous published WGS projects, and relatively poor Internet connectivity.11 Rickettsia typhi is an obligate intracellular, Gram-negative bacterium in the family Rickettsiaceae that causes the disease murine typhus and has a worldwide tropical and subtropical distribution.12,13 The pathogen is transmitted to humans primarily by flea fecal contamination of the bite of the oriental rat flea Xenopsylla cheopis.14 The disease is an important and grossly under-recognized global cause of febrile illness.15 Those affected suffer from high fever, headache, myalgia, arthralgia, nausea, and vomiting and may have a macular rash. Complications are infrequent but include myocarditis, meningoencephalitis, seizures, and renal failure.13,16 When diagnosed, recovery is typically rapid after treatment with doxycycline and mortality is low (around 1–2%) with antibiotic treatment.12,17 Despite its importance as a pathogen, to date just three R. typhi whole genome sequences, with wide geographic and temporal distribution, have been published. The type strain Wilmington was isolated from a patient in North Carolina in 1928, and the sequence was published in 2004.18,19 Two further sequences originating from a patient in Northern Thailand collected in 1965 and the other from a bandicoot rat, Bandicota sp., collected in Burma (now Myanmar) in the 1970s20–22 were published online in 2014 (Table 1). Table 1 Strain information

Background Murine typhus, or infection with Rickettsia typhi, is a global but neglected disease without randomized clinical trials to guide antibiotic therapy. Methods A prospective, open, randomized trial was conducted in nonpregnant, consenting inpatient adults with rapid diagnostic test evidence of uncomplicated murine typhus at 2 hospitals in Vientiane, Laos. Patients were randomized to 7 days (D7) or 3 days (D3) of oral doxycycline or 3 days of oral azithromycin (A3). Primary outcome measures were fever clearance time and frequencies of treatment failure and relapse. Results Between 2004 and 2009, the study enrolled 216 patients (72 per arm); 158 (73.2%) had serology/polymerase chain reaction (PCR)–confirmed murine typhus, and 52 (24.1%) were R. typhi PCR positive. The risk of treatment failure was greater for regimen A3 (22.5%; 16 of 71 patients) than for D3 (4.2%; 3 of 71) or D7 (1.4%; 1 of 71) (P < .001). Among R. typhi PCR–positive patients, the area under the time-temperature curve and the fever clearance time were significantly higher for A3 than for D3 (1.8- and 1.9-fold higher, respectively; P = .005) and D7 (1.5- and 1.6-fold higher; P = .02). No patients returned with PCR-confirmed R. typhi relapse. Conclusion In Lao adults, azithromycin is inferior to doxycycline as oral therapy for uncomplicated murine typhus. For doxycycline, 3- and 7-day regimens have similar efficacy. Azithromycin use in murine typhus should be reconsidered. Investigation of genomic and phenotypic markers of R. typhi azithromycin resistance is needed. Clinical Trial Registration ISRCTN47812566., A clinical trial of murine typhus, or infection with Rickettsia typhi, in Lao adults suggests that azithromycin is inferior to doxycycline for the oral therapy of uncomplicated murine typhus. Three and 7 days of doxycycline have similar efficacy.

The infrastructure challenges and costs of next-generation sequencing have been largely overcome, for many sequencing applications, by Oxford Nanopore Technologies’ portable MinION sequencer. However the question remains open whether MinION-based bacterial whole-genome sequencing (WGS) is by itself sufficient for the accurate assessment of phylogenetic and epidemiological relationships between isolates and whether such tasks can be undertaken in resource-limited settings. To investigate this question, we sequenced the genome of an isolate ofRickettsia typhi, an important and neglected cause of fever across much of the tropics and subtropics, for which only three genomic sequences previously existed. We prepared and sequenced libraries on a MinION in Vientiane, Lao PDR using v9.5 chemistry and in parallel we sequenced the same isolate on the Illumina platform in a genomics laboratory in the UK. The MinION sequence reads yielded a single contiguous assembly, in which the addition of Illumina data revealed 226 base-substitution and 5,856 in/del errors. The combined assembly represents the first complete genome sequence of a humanR. typhiisolate collected in the last 50 years and differed from the genomes of existing strains collected over a 90-year time period at very few sites, and with no re-arrangements. Filtering based on the known error profile of MinION data improved the accuracy of the Nanopore-only assembly. However, the frequency of false-positive errors remained greater than true sequence divergence from recorded sequences. While Nanopore-only sequencing cannot yet recover phylogenetic signal inR. typhi, such an approach may be applicable for more diverse organisms.

In this study, we examined the diagnostic accuracy of the InBios Scrub Typhus Detect IgM enzyme-linked immunosorbent assay (ELISA) and determined the optimal diagnostic optical density (OD) cutoffs for screening and diagnostic applications based on prospectively collected, characterized samples from undifferentiated febrile illness patients in northern Thailand. Direct comparisons with the serological gold standard, indirect immunofluorescence assay (IFA), revealed strong statistical correlation of ELISA OD values and IFA IgM titers. Determination of the optimal ELISA cutoff for seroepidemiology or screening purposes compared to the corresponding IFA reciprocal titer of 400 as previously described for Thailand was 0.60 OD, which corresponded to a sensitivity (Sn) of 84% and a specificity (Sp) of 98%. The diagnostic performance against the improved and more-stringent scrub typhus infection criteria (STIC), correcting for low false-positive IFA titers, resulted in an Sn of 93% and an Sp of 91% at an ELISA cutoff of 0.5 OD. This diagnostic ELISA cutoff corresponds to IFA reciprocal titers of 1,600 to 3,200, which greatly reduces the false-positive rates associated with low-positive IFA titers. These data are in congruence with the recently improved serodiagnostic positivity criteria using the Bayesian latent class modeling approach. In summary, the InBios Scrub Typhus Detect IgM ELISA is affordable and easy-to-use, with adequate diagnostic accuracy for screening and diagnostic purposes, and should be considered an improved alternative to the gold standard IFA for acute diagnosis. For broader application, regional cutoff validation and antigenic composition for consistent diagnostic accuracy should be considered.

The HCV-IRES sequence is vital for both protein translation and genome replication and serves as a potential target for anti-HCV therapy. We constructed a series of anti-HCV group I introns (αHCV-GrpIs) to attack conserved target sites within the HCV IRES. These αHCV-GrpIs were designed to mediate a trans-splicing reaction that replaces the viral RNA genome downstream of the 5′ splice site with a 3′ exon that encodes an apoptosis-inducing gene. Pro-active forms of the apoptosis inducing genes BID, Caspase 3, Caspase 8, or tBax were modified by incorporation of the HCV NS5A/5B cleavage sequence in place of their respective endogenous cleavage sites to ensure that only HCV infected cells would undergo apoptosis following splicing and expression. Huh7.5 cells transfected with each intron were challenged at MOI 0.1 with HCV-Jc1FLAG2 which expresses a Gaussia Luciferase (GLuc) marker. Virus-containing supernatants were then assayed for GLuc expression as a measure of viral replication inhibition. Cellular extracts were analyzed for the presence of correct splice products by RT-PCR and DNA sequencing. We also measured levels of Caspase 3 activity as a means of quantifying apoptotic cell death. Each of these αHCV-GrpI introns was able to correctly splice their 3′ apoptotic exons onto the virus RNA genome at the targeted Uracil, and resulted in greater than 80% suppression of the GLuc marker. A more pronounced suppression effect was observed with TCID 50 virus titrations, which demonstrated that these αHCV-GrpIs were able to suppress viral replication by more than 2 logs, or greater than 99%. Robust activation of the apoptotic factor within the challenged cells was evidenced by a significant increase of Caspase 3 activity upon viral infection compared to non-challenged cells. This novel genetic intervention tool may prove beneficial in certain HCV subjects.

We developed an intradermal (ID) challenge cynomolgus macaque (Macaca fascicularis) model of scrub typhus, the leading cause of treatable undifferentiated febrile illness in tropical Asia, caused by the obligate intracellular bacterium, Orientia tsutsugamushi. A well-characterized animal model is required for the development of clinically relevant diagnostic assays and evaluation of therapeutic agents and candidate vaccines. We investigated scrub typhus disease pathophysiology and evaluated two O. tsutsugamushi 47-kDa, Ag-based candidate vaccines, a DNA plasmid vaccine (pKarp47), and a virus-vectored vaccine (Kp47/47-Venezuelan equine encephalitis virus replicon particle) for safety, immunogenicity, and efficacy against homologous ID challenge with O. tsutsugamushi Karp. Control cynomolgus macaques developed fever, classic eschars, lymphadenopathy, bacteremia, altered liver function, increased WBC counts, pathogen-specific Ab (IgM and IgG), and cell-mediated immune responses. Vaccinated macaques receiving the DNA plasmid pKarp47 vaccine had significantly increased O. tsutsugamushi–specific, IFN-γ–producing PBMCs (p = 0.04), reduced eschar frequency and bacteremia duration (p ≤ 0.01), delayed bacteremia onset (p < 0.05), reduced circulating bacterial biomass (p = 0.01), and greater reduction of liver transaminase levels (p < 0.03) than controls. This study demonstrates a vaccine-induced immune response capable of conferring sterile immunity against high-dose homologous ID challenge of O. tsutsugamushi in a nonhuman primate model, and it provides insight into cell-mediated immune control of O. tsutsugamushi and dissemination dynamics, highlights the importance of bacteremia indices for evaluation of both natural and vaccine-induced immune responses, and importantly, to our knowledge, has determined the first phenotypic correlates of immune protection in scrub typhus. We conclude that this model is suitable for detailed investigations into vaccine-induced immune responses and correlates of immunity for scrub typhus.

In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like the dengue virus RNA genomes. These experiments are performed for initial assessment of HHR catalysis in a cell-free system and have been simplified by the substitution of agarose gel electrophoresis for SDS-PAGE. Substituting mobility assays enables the analysis of ribozymes in a more rapid fashion without radioisotopes. Here we describe the in vitro transcription of an HHR and corresponding target from T7-promoted plasmids into RNA molecules leading to the analysis of HHR activity against the RNA target by in vitro cleavage assays.

Background There is an urgent need to develop rapid and accurate point-of-care (POC) technologies for acute scrub typhus diagnosis in low-resource, primary health care settings to guide clinical therapy. Methodology/Principal Findings In this study we present the clinical evaluation of loop-mediated isothermal PCR assay (LAMP) in the context of a prospective fever study, including 161 patients from scrub typhus-endemic Chiang Rai, northern Thailand. A robust reference comparator set comprising following ‘scrub typhus infection criteria’ (STIC) was used: a) positive cell culture isolate and/or b) an admission IgM titer ≥1∶12,800 using the ‘gold standard’ indirect immunofluorescence assay (IFA) and/or c) a 4-fold rising IFA IgM titer and/or d) a positive result in at least two out of three PCR assays. Compared to the STIC criteria, all PCR assays (including LAMP) demonstrated high specificity ranging from 96–99%, with sensitivities varying from 40% to 56%, similar to the antibody based rapid test, which had a sensitivity of 47% and a specificity of 95%. Conclusions/Significance The diagnostic accuracy of the LAMP assay was similar to realtime and nested conventional PCR assays, but superior to the antibody-based rapid test in the early disease course. The combination of DNA- and antibody-based detection methods increased sensitivity with minimal reduction of specificity, and expanded the timeframe of adequate diagnostic coverage throughout the acute phase of scrub typhus., Author Summary There is an urgent need for alternative diagnostic methods for scrub typhus, but evaluation of these is hampered because the current serological gold standard (IFA) is imperfect. In a study from Thailand, 3 of 20 (15%) patients with fever had a positive Orientia tsutsugamushi PCR result despite negative serology. These findings could reflect potential benefits of the PCR assay in detecting rickettsaemia before antibody responses set in and/or a diagnostic advantage in endemic areas with high background levels of antibody in the population. Serology is complicated by the heterogeneity of strains present in Southeast Asia, but high resource costs and training make realtime PCR assays impractical for many areas where scrub typhus is endemic. This is where the new LAMP methodology has potential: it is inexpensive, simple to perform and requires only a waterbath or simple heating block instead of a thermocycler. In the context of a prospective fever study in a scrub typhus-endemic area in Thailand, the results support the validity of LAMP methodology for the diagnosis of scrub typhus, highlight the difficulties in comparing antibody- with DNA-based methods and also contribute towards understanding the dynamics of bacteraemia in this under recognised and under studied disease.

Outbreaks of Dengue impose a heavy economic burden on developing countries in terms of vector control and human morbidity. Effective vaccines against all four serotypes of Dengue are in development, but population replacement with transgenic vectors unable to transmit the virus might ultimately prove to be an effective approach to disease suppression, or even eradication. A key element of the refractory transgenic vector approach is the development of transgenes that effectively prohibit viral transmission. In this report we test the effectiveness of several hammerhead ribozymes for suppressing DENV in lentivirus-transduced mosquito cells in an attempt to mimic the transgenic use of these effector molecules in mosquitoes. A lentivirus vector that expresses these ribozymes as a fusion RNA molecule using an Ae. aegypti tRNAval promoter and terminating with a 60A tail insures optimal expression, localization, and activity of the hammerhead ribozyme against the DENV genome. Among the 14 hammerhead ribozymes we designed to attack the DENV-2 NGC genome, several appear to be relatively effective in reducing virus production from transduced cells by as much as 2 logs. Among the sequences targeted are 10 that are conserved among all DENV serotype 2 strains. Our results confirm that hammerhead ribozymes can be effective in suppressing DENV in a transgenic approach, and provide an alternative or supplementary approach to proposed siRNA strategies for DENV suppression in transgenic mosquitoes.

Scrub typhus (caused by Orientia tsutsugamushi) and murine typhus (caused by Rickettsia typhi) cause up to 28% of febrile episodes in Thailand and Laos. The current understanding of coagulation and inflammation in the pathogenesis of these clinically very similar vasculotropic diseases is limited. This study compared human in vivo changes in 15 coagulation, inflammation and endothelial activation markers in prospectively collected admission and follow-up samples of 121 patients (55 scrub typhus, 55 murine typhus, and 11 typhus-like illness) and 51 healthy controls from Laos. As compared with controls, all but one of the markers assessed were significantly affected in typhus patients; however, the activation patterns differed significantly between scrub and murine typhus patients. The levels of markers of coagulation activation and all inflammatory cytokines, except for interleukin-12, were significantly higher in patients with scrub typhus than in those with murine typhus. In patients with murine typhus, however, the levels of endothelium-derived markers were significantly higher. Anticoagulant factors were inhibited in both typhus patient groups. This is the first study demonstrating that, in scrub typhus, in vivo coagulation activation is prominent and is related to a strong proinflammatory response, whereas in murine typhus, changes in coagulant and fibrinolytic pathways are suggestive of endothelial cell perturbation. These data suggest that, although late-stage endothelial infection is common in both diseases, the in vivo pathogenic mechanisms of R. typhi and O. tsutsugamushi could differ in the early phase of infection and may contribute to disease differentiation. © 2011 The Authors. Clinical Microbiology and Infection © 2011 European Society of Clinical Microbiology and Infectious Diseases.