• 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

      Carroll, M; Haldenby, S; Rickett, N; Pályi, B; Garcia-Dorival, I; Liu, X; Barker, G; Bore, J; Koundouno, F; Williamson, E; et al. (American Society for Microbiology, 2017-08-23)
      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.
    • Real-Time PCR for the Evaluation of Treatment Response in Clinical Trials of Adult Chronic Chagas Disease: Usefulness of Serial Blood Sampling and qPCR Replicates

      Parrado, R; Ramirez, JC; de la Barra, A; Alonso-Vega, C; Juiz, N; Ortiz, L; Illanes, D; Torrico, F; Gascon, J; Alves, F; et al. (American Society for Microbiology, 2018-12-03)
      This work evaluated a serial blood sampling procedure to enhance the sensitivity of duplex real-time PCR (qPCR) for baseline detection and quantification of parasitic loads and post-treatment identification of failure in the context of clinical trials for treatment of chronic Chagas disease, namely DNDi-CH-E1224-001 (NCT01489228) and MSF-DNDi PCR sampling optimization study (NCT01678599). Patients from Cochabamba (N= 294), Tarija (N= 257), and Aiquile (N= 220) were enrolled. Three serial blood samples were collected at each time-point and qPCR triplicates were tested per sample. The first two samples were collected during the same day and the third one seven days later.A patient was considered PCR positive if at least one qPCR replicate was detectable. Cumulative results of multiple samples and qPCR replicates enhanced the proportion of pre-treatment sample positivity from 54.8 to 76.2%, 59.5 to 77.8%, and 73.5 to 90.2% in Cochabamba, Tarija, and Aiquile cohorts, respectively. This strategy increased the detection of treatment failure from 72.9 to 91.7%, 77.8 to 88.9%, and 42.9 to 69.1% for E1224 low, short, and high dosage regimens, respectively; and from 4.6 to 15.9% and 9.5 to 32.1% for the benznidazole arm in the DNDi-CH-E1224-001 and MSF-DNDi studies, respectively. The addition of the third blood sample and third qPCR replicate in patients with non-detectable PCR results in the first two samples, gave a small, non-statistically significant improvement in qPCR positivity. No change in clinical sensitivity was seen with a blood volume increase from 5 to 10 ml. The monitoring of patients treated with placebo in the DNDi-CH-E1224-001 trial revealed fluctuations in parasitic loads and occasional non-detectable results. In conclusion, serial sampling strategy enhanced PCR sensitivity to detecting treatment failure during follow-up and has the potential for improving recruitment capacity in Chagas disease trials, which require an initial positive qPCR result for patient admission.
    • Transmission of ebola viruses: what we know and what we do not know

      Osterholm, Michael T; Moore, Kristine A; Kelley, Nicholas S; Brosseau, Lisa M; Wong, Gary; Murphy, Frederick A; Peters, Clarence J; LeDuc, James W; Russell, Phillip K; Van Herp, Michel; et al. (American Society for Microbiology, 2015-02-19)
      Available evidence demonstrates that direct patient contact and contact with infectious body fluids are the primary modes for Ebola virus transmission, but this is based on a limited number of studies. Key areas requiring further study include (i) the role of aerosol transmission (either via large droplets or small particles in the vicinity of source patients), (ii) the role of environmental contamination and fomite transmission, (iii) the degree to which minimally or mildly ill persons transmit infection, (iv) how long clinically relevant infectiousness persists, (v) the role that "superspreading events" may play in driving transmission dynamics, (vi) whether strain differences or repeated serial passage in outbreak settings can impact virus transmission, and (vii) what role sylvatic or domestic animals could play in outbreak propagation, particularly during major epidemics such as the 2013-2015 West Africa situation. In this review, we address what we know and what we do not know about Ebola virus transmission. We also hypothesize that Ebola viruses have the potential to be respiratory pathogens with primary respiratory spread.