A robust host-response-based signature distinguishes bacterial and viral infections across diverse global populations.

Limited sensitivity and specificity of current diagnostics lead to the erroneous prescription of antibiotics. Host-response-based diagnostics could address these challenges. However, using 4,200 samples across 69 blood transcriptome datasets from 20 countries from patients with bacterial or viral infections representing a broad spectrum of biological, clinical, and technical heterogeneity, we show current host-response-based gene signatures have lower accuracy to distinguish intracellular bacterial infections from viral infections than extracellular bacterial infections. Using these 69 datasets, we identify an 8-gene signature to distinguish intracellular or extracellular bacterial infections from viral infections with an area under the receiver operating characteristic curve (AUROC) > 0.91 (85.9% specificity and 90.2% sensitivity). In prospective cohorts from Nepal and Laos, the 8-gene classifier distinguished bacterial infections from viral infections with an AUROC of 0.94 (87.9% specificity and 91% sensitivity). The 8-gene signature meets the target product profile proposed by the World Health Organization and others for distinguishing bacterial and viral infections.

Zoonotic Pathogens in Wildlife Traded in Markets for Human Consumption, Laos.

We tested animals from wildlife trade sites in Laos for the presence of zoonotic pathogens. Leptospira spp. were the most frequently detected infectious agents, found in 20.1% of animals. Rickettsia typhi and R. felis were also detected. These findings suggest a substantial risk for exposure through handling and consumption of wild animal meat.

Oxford Nanopore MinION Sequencing Enables Rapid Whole Genome Assembly of Rickettsia typhi in a Resource-Limited Setting.

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 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 of Rickettsia 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 United Kingdom. The MinION sequence reads yielded a single contiguous assembly, in which the addition of Illumina data revealed 226 base-substitution and 5,856 indel errors. The combined assembly represents the first complete genome sequence of a human R. typhi isolate 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, with no rearrangements. 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. Although nanopore-only sequencing cannot yet recover phylogenetic signals in R. typhi, such an approach may be applicable for more diverse organisms.

A Prospective, Open-label, Randomized Trial of Doxycycline Versus Azithromycin for the Treatment of Uncomplicated Murine Typhus.

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.

Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand.

BACKGROUND: Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. CONCLUSIONS/SIGNIFICANCE: Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes.

Diagnostic Accuracy of the InBios Scrub Typhus Detect Enzyme-Linked Immunoassay for the Detection of IgM Antibodies in Northern Thailand.

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.

Trans-splicing group I intron targeting hepatitis C virus IRES mediates cell death upon viral infection in Huh7.5 cells.

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 TCID50 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.

A nonhuman primate scrub typhus model: protective immune responses induced by pKarp47 DNA vaccination in cynomolgus macaques.

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.

Design and analysis of hammerhead ribozyme activity against an artificial gene target.

In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like dengue virus RNA. 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.

Diagnostic accuracy of a loop-mediated isothermal PCR assay for detection of Orientia tsutsugamushi during acute Scrub Typhus infection.

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.

Effective suppression of Dengue fever virus in mosquito cell cultures using retroviral transduction of hammerhead ribozymes targeting the viral genome.

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 tRNA(val) 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.