Deep Sequencing of RNA from Blood and Oral Swab Samples Reveals the Presence of Nucleic Acid from a Number of Pathogens in Patients with Acute Ebola Virus Disease and Is Consistent with Bacterial Translocation across the Gut.

In this study, samples from the 2013-2016 West African Ebola virus outbreak from patients in Guinea with Ebola virus disease (EVD) were analyzed to discover and classify what other pathogens were present. Throat swabs were taken from deceased EVD patients, and peripheral blood samples were analyzed that had been taken from patients when they presented at the treatment center with acute illness. High-throughput RNA sequencing (RNA-seq) and bioinformatics were used to identify the potential microorganisms. This approach confirmed Ebola virus (EBOV) in all samples from patients diagnosed as acute positive for the virus by quantitative reverse transcription-PCR in deployed field laboratories. Nucleic acid mapping to Plasmodium was also used on the patient samples, confirming results obtained with an antigen-based rapid diagnostic test (RDT) conducted in the field laboratories. The data suggested that a high Plasmodium load, as determined by sequence read depth, was associated with mortality and influenced the host response, whereas a lower parasite load did not appear to affect outcome. The identifications of selected bacteria from throat swabs via RNA-seq were confirmed by culture. The data indicated that the potential pathogens identified in the blood samples were associated with translocation from the gut, suggesting the presence of bacteremia, which transcriptome data suggested may induce or aggravate the acute-phase response observed during EVD. Transcripts mapping to different viruses were also identified, including those indicative of lytic infections. The development of high-resolution analysis of samples from patients with EVD will help inform care pathways and the most appropriate general antimicrobial therapy to be used in a resource-poor setting. IMPORTANCE Our results highlight the identification of an array of pathogens in the blood of patients with Ebola virus disease (EVD). This has not been done before, and the data have important implications for the treatment of patients with EVD, particularly considering antibiotic stewardship. We show that EVD patients who were also infected with Plasmodium, particularly at higher loads, had more adverse outcomes than patients with lower levels of Plasmodium. However, the presence of Plasmodium did not influence the innate immune response, and it is likely that the presence of EBOV dominated this response. Several viruses other than EBOV were identified, and bacteria associated with sepsis were also identified. These findings were indicative of bacterial translocation across the gut during the acute phase of EVD

Language endangerment and language documentation in Africa

Non peer reviewe

Temporal and spatial analysis of the 2014-2015 Ebola virus outbreak in West Africa

West Africa is currently witnessing the most extensive Ebola virus (EBOV) outbreak so far recorded. Until now, there have been 27,013 reported cases and 11,134 deaths. The origin of the virus is thought to have been a zoonotic transmission from a bat to a two-year-old boy in December 2013 (ref. 2). From this index case the virus was spread by human-to-human contact throughout Guinea, Sierra Leone and Liberia. However, the origin of the particular virus in each country and time of transmission is not known and currently relies on epidemiological analysis, which may be unreliable owing to the difficulties of obtaining patient information. Here we trace the genetic evolution of EBOV in the current outbreak that has resulted in multiple lineages. Deep sequencing of 179 patient samples processed by the European Mobile Laboratory, the first diagnostics unit to be deployed to the epicentre of the outbreak in Guinea, reveals an epidemiological and evolutionary history of the epidemic from March 2014 to January 2015. Analysis of EBOV genome evolution has also benefited from a similar sequencing effort of patient samples from Sierra Leone. Our results confirm that the EBOV from Guinea moved into Sierra Leone, most likely in April or early May. The viruses of the Guinea/Sierra Leone lineage mixed around June/July 2014. Viral sequences covering August, September and October 2014 indicate that this lineage evolved independently within Guinea. These data can be used in conjunction with epidemiological information to test retrospectively the effectiveness of control measures, and provides an unprecedented window into the evolution of an ongoing viral haemorrhagic fever outbreak.status: publishe

Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Double Labeling of Oligonucleotide Probes for Fluorescence In Situ Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility ▿ †

Fluorescence in situ hybridization (FISH) with singly labeled rRNA-targeted oligonucleotide probes is widely applied for direct identification of microbes in the environment or in clinical specimens. Here we show that a replacement of singly labeled oligonucleotide probes with 5′-, 3′-doubly labeled probes at least doubles FISH signal intensity without causing specificity problems. Furthermore, Cy3-doubly labeled probes strongly increase in situ accessibility of rRNA target sites and thus provide more flexibility for probe design

Regional variations in waste minimization in England: Challenges and issues for policy development

Exploring regional variations in waste minimization in England. This is vital to underpin coming waste strategies. There are a number of key challenges and issues for policy development that must be addressed urgently

Pulse-Controlled Amplification-A new powerful tool for on-site diagnostics under resource limited conditions.

BackgroundMolecular diagnostics has become essential in the identification of many infectious and neglected diseases, and the detection of nucleic acids often serves as the gold standard technique for most infectious agents. However, established techniques like polymerase chain reaction (PCR) are time-consuming laboratory-bound techniques while rapid tests such as Lateral Flow Immunochromatographic tests often lack the required sensitivity and/or specificity.Methods/principle findingsHere we present an affordable, highly mobile alternative method for the rapid identification of infectious agents using pulse-controlled amplification (PCA). PCA is a next generation nucleic acid amplification technology that uses rapid energy pulses to heat microcyclers (micro-scale metal heating elements embedded directly in the amplification reaction) for a few microseconds, thus only heating a small fraction of the reaction volume. The heated microcyclers cool off nearly instantaneously, resulting in ultra-fast heating and cooling cycles during which classic amplification of a target sequence takes place. This reduces the overall amplification time by a factor of up to 10, enabling a sample-to-result workflow in just 15 minutes, while running on a small and portable prototype device. In this proof of principle study, we designed a PCA-assay for the detection of Yersinia pestis to demonstrate the efficacy of this technology. The observed detection limits were 434 copies per reaction (purified DNA) and 35 cells per reaction (crude sample) respectively of Yersinia pestis.Conclusions/significancePCA offers fast and decentralized molecular diagnostics and is applicable whenever rapid, on-site detection of infectious agents is needed, even under resource limited conditions. It combines the sensitivity and specificity of PCR with the rapidness and simplicity of hitherto existing rapid tests

16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales".

For simultaneous identification of members of the betaproteobacterial order “Rhodocyclales” in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the “Rhodocyclales.” The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent “Rhodocyclales” diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed “Rhodocyclales”-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of “Rhodocyclales” populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology