Comparison of Illumina and Oxford Nanopore Sequencing Technologies for Pathogen Detection from Clinical Matrices Using Molecular Inversion Probes.

Next-generation sequencing is rapidly finding footholds in numerous microbiological fields, including infectious disease diagnostics. Here, we describe a molecular inversion probe panel for the identification of bacterial, viral, and parasitic pathogens. We describe the ability of Illumina and Oxford Nanopore Technologies (ONT) to sequence small amplicons originating from this panel for the identification of pathogens in complex matrices. The panel correctly classified 31 bacterial pathogens directly from positive blood culture bottles with a genus-level concordance of 96.7% and 90.3% on the Illumina and ONT platforms, respectively. Both sequencing platforms detected 18 viral and parasitic organisms directly from mock clinical samples of plasma and whole blood at concentrations of 104 PFU/mL with few exceptions. In general, Illumina sequencing exhibited greater read counts with lower percent mapped reads; however, this resulted in no effect on limits of detection compared with ONT sequencing. Mock clinical evaluation of the probe panel on the Illumina and ONT platforms resulted in positive predictive values of 0.91 and 0.88 and negative predictive values of 1 and 1 from de-identified human chikungunya virus samples compared with gold standard quantitative RT-PCR. Overall, these data show that molecular inversion probes are an adaptable technology capable of pathogen detection from complex sample matrices on current next-generation sequencing platforms.

Host response transcriptomic analysis of Crimean-Congo hemorrhagic fever pathogenesis in the cynomolgus macaque model.

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne RNA virus prevalent in Asia, Europe, and Africa, and can cause a hemorrhagic disease (CCHF) in humans with mortality rates as high as 60%. A general lack of both effective medical countermeasures and a comprehensive understanding of disease pathogenesis is partly driven by an historical lack of viable CCHF animal models. Recently, a cynomolgous macaque model of CCHF disease was developed. Here, we document the targeted transcriptomic response of non-human primates (NHP) to two different CCHFV strains; Afghan09-2990 and Kosova Hoti that both yielded a mild CCHF disease state. We utilized a targeted gene panel to elucidate the transcriptomic changes occurring in NHP whole blood during CCHFV infection; a first for any primate species. We show numerous upregulated genes starting at 1 day post-challenge through 14 days post-challenge. Early gene changes fell predominantly in the interferon stimulated gene family with later gene changes coinciding with an adaptive immune response to the virus. There are subtle differences between viral strains, namely duration of the differentially expressed gene response and biological pathways enriched. After recovery, NHPs showed no lasting transcriptomic changes at the end of sample collection.

Transcriptomic Analysis Reveals Host miRNAs Correlated with Immune Gene Dysregulation during Fatal Disease Progression in the Ebola Virus Cynomolgus Macaque Disease Model.

Ebola virus is a continuing threat to human populations, causing a virulent hemorrhagic fever disease characterized by dysregulation of both the innate and adaptive host immune responses. Severe cases are distinguished by an early, elevated pro-inflammatory response followed by a pronounced lymphopenia with B and T cells unable to mount an effective anti-viral response. The precise mechanisms underlying the dysregulation of the host immune system are poorly understood. In recent years, focus on host-derived miRNAs showed these molecules to play an important role in the host gene regulation arsenal. Here, we describe an investigation of RNA biomarkers in the fatal Ebola virus disease (EVD) cynomolgus macaque model. We monitored both host mRNA and miRNA responses in whole blood longitudinally over the disease course in these non-human primates (NHPs). Analysis of the interactions between these classes of RNAs revealed several miRNA markers significantly correlated with downregulation of genes; specifically, the analysis revealed those involved in dysregulated immune pathways associated with EVD. In particular, we noted strong interactions between the miRNAs hsa-miR-122-5p and hsa-miR-125b-5p with immunological genes regulating both B and T-cell activation. This promising set of biomarkers will be useful in future studies of severe EVD pathogenesis in both NHPs and humans and may serve as potential prognostic targets.

Diagnostic targETEd seQuencing adjudicaTion (DETEQT): Algorithms for Adjudicating Targeted Infectious Disease Next-Generation Sequencing Panels.

Next-generation sequencing (NGS) for infectious disease diagnostics is a relatively new and underdeveloped concept. If this technology is to become a regulatory-grade clinical diagnostic, standardization in the form of locked-down assays and firmly established underlying processes is necessary. Targeted sequencing, specifically by amplification of genomic signatures, has the potential to bridge the gap between PCR- and NGS-based diagnostics; however, existing NGS assay panels lack validated analytical techniques to adjudicate high background and error-prone NGS data. Herein, we present the Diagnostic targETEd seQuencing adjudicaTion (DETEQT) software, consisting of an intuitive bioinformatics pipeline entailing a set of algorithms to translate raw sequencing data into positive, negative, and indeterminate diagnostic determinations. After basic read filtering and mapping, the software compares abundance and quality metrics against heuristic and fixed thresholds. A novel generalized quality function provides an amalgamated quality score for the match between sequence reads of an assay and panel targets, rather than considering each component factor independently. When evaluated against numerous assay samples and parameters (mock clinical, human, and nonhuman primate clinical data sets; diverse amplification strategies; downstream applications; and sequence platforms), DETEQT demonstrated improved rejection of false positives and accuracies >95%. Finally, DETEQT was implemented in the user-friendly Empowering the Development of Genomics Expertise (EDGE) bioinformatics platform, providing a complete, end-to-end solution that can be operated by nonexperts in a clinical laboratory setting.

Rapid antibiotic susceptibility testing from blood culture bottles with species agnostic real-time polymerase chain reaction.

Development and implementation of rapid antimicrobial susceptibility testing is critical for guiding patient care and improving clinical outcomes, especially in cases of sepsis. One approach to reduce the time-to-answer for antimicrobial susceptibility is monitoring the inhibition of DNA production, as differences in DNA concentrations are more quickly impacted compared to optical density changes in traditional antimicrobial susceptibility testing. Here, we use real-time PCR to rapidly determine antimicrobial susceptibility after short incubations with antibiotic. Application of this assay to a collection of 144 isolates in mock blood culture, covering medically relevant pathogens displaying high rates of resistance, provided susceptibility data in under 4 hours. This assay provided categorical agreement with a reference method in 96.3% of cases across all species. Sequencing of a subset of PCR amplicons showed accurate genus level identification. Overall, implementation of this method could provide accurate susceptibility results with a reduced time-to-answer for a number of medically relevant bacteria commonly isolated from blood culture.

Immune activation alters cellular and humoral responses to yellow fever 17D vaccine.

BACKGROUND: Defining the parameters that modulate vaccine responses in African populations will be imperative to design effective vaccines for protection against HIV, malaria, tuberculosis, and dengue virus infections. This study aimed to evaluate the contribution of the patient-specific immune microenvironment to the response to the licensed yellow fever vaccine 17D (YF-17D) in an African cohort. METHODS: We compared responses to YF-17D in 50 volunteers in Entebbe, Uganda, and 50 volunteers in Lausanne, Switzerland. We measured the CD8+ T cell and B cell responses induced by YF-17D and correlated them with immune parameters analyzed by flow cytometry prior to vaccination. RESULTS: We showed that YF-17D-induced CD8+ T cell and B cell responses were substantially lower in immunized individuals from Entebbe compared with immunized individuals from Lausanne. The impaired vaccine response in the Entebbe cohort associated with reduced YF-17D replication. Prior to vaccination, we observed higher frequencies of exhausted and activated NK cells, differentiated T and B cell subsets and proinflammatory monocytes, suggesting an activated immune microenvironment in the Entebbe volunteers. Interestingly, activation of CD8+ T cells and B cells as well as proinflammatory monocytes at baseline negatively correlated with YF-17D-neutralizing antibody titers after vaccination. Additionally, memory T and B cell responses in preimmunized volunteers exhibited reduced persistence in the Entebbe cohort but were boosted by a second vaccination. CONCLUSION: Together, these results demonstrate that an activated immune microenvironment prior to vaccination impedes efficacy of the YF-17D vaccine in an African cohort and suggest that vaccine regimens may need to be boosted in African populations to achieve efficient immunity. TRIAL REGISTRATION: Registration is not required for observational studies. FUNDING: This study was funded by Canada's Global Health Research Initiative, Defense Threat Reduction Agency, National Institute of Allergy and Infectious Diseases, Bill & Melinda Gates Foundation, and United States Agency for International Development.

Origin of the dengue virus outbreak in Martin County, Florida, USA 2013.

After a 75-year absence from Florida, substantial local transmission of dengue virus (DENV) occurred in Key West, Monroe County, Florida in 2009 and continued in 2010. The outbreak culminated in 85 reported cases. In 2011 and 2012, only isolated cases of local DENV transmission were reported in Florida, none were reported in Key West. In 2013, a new outbreak occurred, but this time in Martin County about 275 miles North of Key West with 22 reported cases. As the Key West and Martin County outbreaks involved DENV serotype 1 (DENV-1), we wanted to investigate whether the same strain or a different strain of DENV was responsible for the outbreaks. In this study, we report the sequence and phylogenetic analysis of the E generegion from a patient diagnosed with dengue in Martin County. Our results indicate that the 2013 Martin County DENV-1 strain is distinct from the 2009-2010 Key West DENV-1 and that it is most closely related to viruses from a recent expansion of South American DENV-1 strains into the Caribbean. We conclude that the 2013 Martin County outbreak was the result of a new introduction of DENV-1 in Florida.

Mechanistic study of broadly neutralizing human monoclonal antibodies against dengue virus that target the fusion loop.

There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies.