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Comparative Study
. 2004 Apr;78(8):4330-41.
doi: 10.1128/jvi.78.8.4330-4341.2004.

Rapid diagnosis of Ebola hemorrhagic fever by reverse transcription-PCR in an outbreak setting and assessment of patient viral load as a predictor of outcome

Jonathan S Towner  1 , Pierre E Rollin, Daniel G Bausch, Anthony Sanchez, Sharon M Crary, Martin Vincent, William F Lee, Christina F Spiropoulou, Thomas G Ksiazek, Mathew Lukwiya, Felix Kaducu, Robert Downing, Stuart T Nichol
Affiliations
Comparative Study

Rapid diagnosis of Ebola hemorrhagic fever by reverse transcription-PCR in an outbreak setting and assessment of patient viral load as a predictor of outcome

Jonathan S Towner et al. J Virol. 2004 Apr.

Abstract

The largest outbreak on record of Ebola hemorrhagic fever (EHF) occurred in Uganda from August 2000 to January 2001. The outbreak was centered in the Gulu district of northern Uganda, with secondary transmission to other districts. After the initial diagnosis of Sudan ebolavirus by the National Institute for Virology in Johannesburg, South Africa, a temporary diagnostic laboratory was established within the Gulu district at St. Mary's Lacor Hospital. The laboratory used antigen capture and reverse transcription-PCR (RT-PCR) to diagnose Sudan ebolavirus infection in suspect patients. The RT-PCR and antigen-capture diagnostic assays proved very effective for detecting ebolavirus in patient serum, plasma, and whole blood. In samples collected very early in the course of infection, the RT-PCR assay could detect ebolavirus 24 to 48 h prior to detection by antigen capture. More than 1,000 blood samples were collected, with multiple samples obtained from many patients throughout the course of infection. Real-time quantitative RT-PCR was used to determine the viral load in multiple samples from patients with fatal and nonfatal cases, and these data were correlated with the disease outcome. RNA copy levels in patients who died averaged 2 log(10) higher than those in patients who survived. Using clinical material from multiple EHF patients, we sequenced the variable region of the glycoprotein. This Sudan ebolavirus strain was not derived from either the earlier Boniface (1976) or Maleo (1979) strain, but it shares a common ancestor with both. Furthermore, both sequence and epidemiologic data are consistent with the outbreak having originated from a single introduction into the human population.

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Figures

FIG. 1.
FIG. 1.
Target specificity of NP primer-probe set designed for the Gulu strain of Sudan ebolavirus. (A) In vitro-transcribed genomic- or antigenomic-sense NP RNA was serially diluted and amplified by using either positive- or negative-sense primers during the RT step. (B) In vitro-transcribed genomic-sense NP RNA was serially diluted and tested either alone or in the presence of RNA isolated from serum or whole blood of an uninfected person.
FIG. 2.
FIG. 2.
Representative viral load profiles of EHF patients as determined by Q-RT-PCR analysis and compared with antigen-capture and IgG levels determined for the same samples. (A) Fatal case profiles. (B) Nonfatal case profiles.
FIG. 3.
FIG. 3.
Summary of RNA copy and antigen levels of EHF cases with fatal and nonfatal outcomes. Each bar represents the arithmetic mean value, and the error bars represent 1 standard error of the mean for each time point. (A) Mean log10 RNA copies per milliliter of serum from 18 survivors and 27 nonsurvivors. The threshold of detection was 6,200 RNA copies per ml of serum and was the lowest level detected in any of the patients analyzed. For the purposes of the graph, this value was assigned to samples of laboratory-confirmed patients that had Ct values of 40. (B) Mean antigen levels determined in duplicate aliquots of those described in panel A. The positive threshold was 0.45 adjusted ODsum units at 410 nm. For samples to be considered positive, they must have had a titer of at least 1:16 and an ODsum of >0.45. (C) Mean antigen levels determined for the specimens described in panel B combined with antigen levels measured in samples from an additional 35 survivors and 62 nonsurvivors that were not tested by Q-RT-PCR (total = 89 fatal and 53 nonfatal cases).
FIG. 4.
FIG. 4.
Comparison between standard single-round and nested RT-PCR and one-step and two-step real-time Q-RT-PCR. All cDNA reactions were programmed with 1 μl of total RNA and were analyzed side-by-side in the indicated RT-PCR assays. (A) Summary chart of RNA dilutions and the subsequent classification (+ or −) by each assay. For the Q-RT-PCR assays, the corresponding triplicate Ct values associated with each RNA dilution are shown. For a result in the Q-RT-PCR assay to be considered positive, at least two of the three triplicate samples had to register a Ct of <40. (B) One percent agarose gel showing the amplification products present in 5 μl of reaction mix for each of the RNA dilutions after standard single-round and nested RT-PCR. The corresponding sizes of the expected DNA amplicons are 185 and 150 nucleotides, respectively.
FIG. 5.
FIG. 5.
Summary of glycoprotein variable region nucleotide sequence analysis (GenBank accession number AY344234). (A) Nucleotides 821 to 1696 (nucleotide numbering of Sudan ebolavirus, Maleo strain [GenBank accession number U23069]) were determined for five EHF patients representing (i) all three geographic distributions of the EHF outbreak within Uganda, (ii) cases from the beginning, middle, and end of the outbreak, and (iii) fatal and nonfatal outcomes. (B) Maximum parsimony analysis of nucleotide sequence differences among the GP variable regions (analogous to nucleotides 821 to 1696 of Sudan ebolavirus, Maleo strain) of representative filoviruses was performed by using a heuristic search option and a 2:1 weighting of transversions over transitions. Bootstrap analysis was conducted with 500 pseudoreplicates of the data set, and values above 50% are shown at branch points. Horizontal distances represent nucleotide step differences (see bar scale), while vertical branches are for visual clarity only.
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