Article Figures & Data
Figures
- FIG 1
Mean time between symptom onset and sample procurement for two of the three different patient categories, hospitalized survivors and hospitalized fatalities.
- FIG 2
Illustrative examples for the total number of reads mapped to species identified in the blood samples from patients with acute EVD.
- FIG 3
Heat map showing the bacterial species (y axis) identified by mapping to nucleic acids by comparing all patient cohorts who had a blood sample taken when they had acute EVD. The x axis shows individual patients from the convalescent control group (green) (n = 16), hospitalized survivors group (yellow) (n = 44), and hospitalized fatalities group (orange) (n = 118); data for these groups are also separated by vertical lines. The colors represent the arbitrary read depth of the sequence, with yellow indicating a greater sequence read depth.
- FIG 4
Heat map showing the bacterial species (y axis) identified mapping to nucleic acids in a swab sample taken from an individual patient. All patients in this category were deceased at the time of sampling (n = 24). The color represents the arbitrary read depth of the sequence, with yellow indicating a greater sequence read depth.
- FIG 5
Relative abundance of transcripts mapping to host transcripts associated with pathways involved in the acute-phase response, blood clotting, innate immune response, and inflammation in patients with EVD (positive by RNA-seq and qRT-PCR) who were confirmed positive (n = 22; gray) or negative (n = 9; black) for at least one bacterial coinfection (by RNA-seq). Relative gene expression levels were measured as fragments per kilobase of transcript per million mapped reads (FPKM). Each of the graphs presented is based on the average FPKM values. Statistical analysis was performed using a two-way ANOVA with Sidak’s multiple-comparisons adjustment (Prism 7; GraphPad, CA).
- FIG 6
Effect of an increased number of reads mapping to P. falciparum. The green line, associated with the right-hand y axis, shows the proportion of patients who tested negative for malaria according to the RDT but for whom reads to P. falciparum were detected during RNA-seq analysis. The red line, also associated with the right-hand y axis, shows the patient fatality rate according to different levels of reads to P. falciparum. The left-hand y axis shows the abundance of patients in each bin, i.e., the number of individuals who were grouped into each category. The graph was created using Prism (GraphPad, CA).
- FIG 7
Relative abundance of transcripts mapping to host transcripts associated with malaria and the innate immune response in patients with EVD who were confirmed positive (gray; n = 10) or negative (black; n = 13) for P. falciparum (by RNA-seq and RDT). Relative gene expression levels were measured as the fragments per kilobase of transcript per million mapped reads (FPKM). Each set of graphs (left and right) is based on average FPKM values. Two analyses of the data are presented, and means and standard deviations (left) or medians and total ranges (right) are shown.
- FIG 8
Relative abundance of transcripts mapping to host transcripts associated with coagulation in patients with EVD and confirmed positive (gray; n = 10) or negative (black; n = 12) for P. falciparum (by RNA-seq and RDT). Relative gene expression levels were measured as fragments per kilobase of transcript per million mapped reads (FPKM). The graphs are based on average FPKM values. Two analyses of the data are presented, showing the means and standard deviations (left) and medians and total ranges (right).
- FIG 9
The presence of nucleic acids from certain viruses varies with patient age. Patients were grouped according to their immunological maturity (≤1 year [n = 8], 1.5 to 4 years [n = 13], 5 to 45 years [n = 120], and 46+ years [n = 39]), and the proportions of each group with reads mapping to P. falciparum, HHV4, and GBV-C are shown.
Tables
- TABLE 1
Summary details of patients from which RNA-seq data were selected for this studya
Patient trait Community deaths Hospitalized fatalities Hospitalized survivors Convalescent controls n 24 118 44 16 % male 29.1 40.1 29.5 93.75 Age range 1–70 yr 2 mo–80 yr 10 mo–68 yr 18–40 yr Mean age (yrs) 33.35 28.44 33.10 30 EBOV CT Range 12.37–31.85 12.08–26.55 15.77–31.60 NA Mean 18.81 17.23 21.67 NA ↵a Samples with similar threshold cycle (CT) values were chosen, as this would indicate similar viral loads. It was not possible to directly correlate CT values to viral load. NA, not applicable.
- TABLE 2
Exemplary data on the variety of microorganisms identified in pooled RNA-seq data from 179 patients positive for EBOV based on qRT-PCR
Organism Protein % identity Alignment E value Escherichia coli G58-1 Glycosyl transferase family 2 100 583 0.00E+0 Lactobacillus fermentum DNA-directed RNA polymerase subunit beta 100 722 0.00E+0 Wohlfahrtiimonas chitiniclastica Hypothetical protein, partial 100 583 0.00E+0 Plasmodium falciparum UGT5.1 Hypothetical protein C923_00328 100 511 4.50E−298 Candida albicans WO-1 Acetyl coenzyme A carboxylase 100 504 2.50E−293 Streptococcus pneumoniae EU-NP04 Transposase DDE domain protein 100 493 2.90E−292 Myroides spp. GMP synthase 100 508 3.20E−292 Haemophilus influenzae Phosphoribosylformylglycinamidine synthase 100 500 1.80E−289 Acinetobacter baumannii AB307-0294 2-Isopropylmalate synthase 100 479 1.20E−276 Providencia rettgeri DSM 1131 DNA polymerase III subunit alpha 100 483 9.70E−276 Plasmodium falciparum Vietnam Oak-Knoll Hypothetical protein PFFVO_04502 100 476 2.20E−270 Ureaplasma urealyticum Cell division protein FtsH 100 478 8.20E−264 Lactobacillus fermentum 6-Phosphogluconate dehydrogenase 100 457 3.70E−260 Plasmodium falciparum Tanzania (2000708) Hypothetical protein PFTANZ_03315 100 450 5.70E−260 Streptococcus sanguinis Collagen-binding protein 100 439 2.80E−251 Acinetobacter baumannii Putrescine/spermidine ABC transporter 100 444 5.10E−251 Haemophilus haemolyticus Catalase 100 415 8.40E−250 Plasmodium falciparum 3D7 Elongation factor 1 alpha 100 433 4.60E−249 Plasmodium falciparum MaliPS096_E11 Histidine-tRNA ligase 100 403 1.10E−235 Myroides spp. Multispecies collagenase 100 413 6.20E−232 Plasmodium falciparum Tanzania (2000708) ATP-dependent protease HslVU, ATPase subunit 100 410 2.10E−229 Campylobacter spp. Multispecies GTP-binding protein 100 407 5.10E−228 Streptococcus parasanguinis Amino acid transporter 100 402 8.80E−228 Haemophilus parainfluenzae l,d-Transpeptidase 100 393 3.80E−224 Escherichia coli Leucyl-tRNA synthetase 100 383 9.40E−224 Myroides spp. Diaminopimelate decarboxylase 100 388 9.30E−223 Myroides odoratimimus TonB-dependent receptor 100 389 1.80E−222 - TABLE 3
Virus sequences identified in pooled RNA-seq data from 179 patients positive for EBOV based on qRT-PCR and clinical diagnosis
Virus Protein % identity Amino acid length E value EBOV L protein 96.8 2,212 0.00E+0 GB-C Polyprotein 99.1 328 1.50E−190 HHV-4 BALF2 100 100 1.9E−51 LF2 protein 100 166 4.9E−91 BALF5 98.8 172 4.4E−88 BALF4 100 257 6.6E−142 DNA polymerase catalytic subunit 99.2 131 2.3E−71 BHRF1 100 121 7.4E-66 BARF0 99.2 120 6.5E−62 Early antigen D 100 118 2E−60 gp110 precursor 100 95 1.3E−49 A73 protein 100 94 6.1E−50 LF1 98.9 92 1.1E−48 Putative BHLF1 protein 98.7 75 4.8E−42 BFRF1 100 71 4.1E−32 BMRF2 100 68 7.6E−31 dUTPase 98.5 68 3E−32 BFRF2 100 67 4.2E−32 LF3 protein 100 45 0.0000000000000002 K15 100 37 0.00000000000056 HHV-5 (cytomegalovirus) IRL4 100 107 3.1E−53 IRL3 94.1 101 1.20E−46 IRL7 100 82 1.60E−37 RL5A 63.6 66 9.30E−15 IRL5 55.1 69 3.50E−12 UL109 68.1 47 2.10E−10 Hepatitis A virus Polyprotein 100 91 5.5E−45 Hepatitis B virus Polymerase 92.8 503 3.30E−278 Pepino mosaic virus Replicase 99.4 163 4.60E−87 Sewage-associated gemycircularvirus-4 Replication-associated protein 100 105 3.60E−57 Rotavirus A VP6 98.8 86 8.1E−41 VP1 100 75 1.1E−33 NSP3 169 251 1.5E−21 Papillomavirus Minor structural protein- interacting protein 100 56 3.40E−24 L2 97.2 72 1.50E−34 Regulatory protein 98.5 67 5E−30 Torque Teno virus 20 Unnamed protein product 100 32 9.10E−9 Tobacco mosaic virus Replicase 97.2 1,521 0.00E+0 Capsid protein 100 102 7.2E−49 126-kDa protein 99 314 2.30E−181 Movement protein 97.4 268 8.60E−145 - TABLE 4
Details of bacterial isolates from throat swab samples identified by mass spectrometry approaches
EBOV CT Age Sex MALDI-TOF result(s)a 24 22 yr Male Proteus mirabilis (2.537) 19 33 yr Female Rothia mucilaginosa (2.181) 25 60 yr NDb Proteus mirabilis (2.427) 14 4 mo Male Staphylococcus aureus (2.378) Acinetobacter baylyi (2.054) 26 63 yr Male Streptococcus salivarius (2.117) Candida albicans (2.141)
TABLE S1
Evidence of genetic material belonging to other pathogens. Download TABLE S1, XLS file, 12.5 MB.
Copyright © 2017 Carroll et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
FIG S1
Phylogenetic tree showing alveolates identified through Diamond BLASTX querying of Trinity-assembled contigs against the nonredundant NCBI database. An E value cutoff of 1 × 10−10 was applied with no additional filtering. Plasmodium species are indicated with red branches, and yellow and red bars indicate the relative abundance levels of sequences associated with species, based on the length of BLAST hits; yellow branches represent log values and red branches represent linear values. Many of the BLAST hits to species with apparently very low hit lengths may have been artifactual but have been retained to provide a contextual background. Download FIG S1, PDF file, 0.4 MB.
Copyright © 2017 Carroll et al.
This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.
