Chan, PKS

Garson, JA

Grant, PR

Sung, JJY

Tam, JS

Tedder, RS

English

UCL Discovery

The new england journal of medicine2468 n engl j med 349;25 www.nejm.org december 18, 2003Detection of SARS Coronavirus in Plasma by Real-Time RT-PCRto the editor: The identification and sequencingof a novel coronavirus1 associated with the recentlydescribed severe acute respiratory syndrome (SARS)2have permitted the development of antibody-basedand genome-based tests for the infection.3,4 Al-though antibody seroconversion provides reliableproof of infection, it is not suitable for early diagno-sis. Techniques for genome detection have the po-tential to provide earlier diagnosis, but the sensitiv-ity of genome-based tests of respiratory samplessuch as nasal and throat swabs is likely to be low inpatients presenting early with fever alone, beforethe development of respiratory symptoms. We there-fore investigated the potential of genome detectionin blood to provide a diagnosis during this very earlyphase.A total of 65 plasma samples obtained within 10days after the onset of fever were available from 26patients with SARS at the Prince of Wales Hospital,Hong Kong. All patients met the World Health Or-ganization’s case definition and had seroconver-sion or a fourfold increase in antibody to the SARS-associated coronavirus (SARS-CoV), as measured byimmunofluorescence. Viral RNA was extracted andamplified by means of a nested reverse-transcrip-tase polymerase chain reaction (RT-PCR), with theuse of both qualitative and quantitative real-timeassays (ABI Prism 7000 system) and the primer setsdescribed by Drosten et al.4 Full details of the assayprotocols are available as Supplementary Appendix1 with the full text of this letter at www.nejm.org.The rate of detection of SARS-CoV RNA in the24 patients tested within three days after the onsetof fever was 79 percent (detection in 19 of the 24patients). Between 3 and 10 days after the onset offever, no additional patients with viremia were iden-tified with the use of either the qualitative or thequantitative assay (Fig. 1A). In the 19 patients inwhom the viral genome could be quantified on oneor more occasions, the plasma viremia level roseearly and was maximal at around day 4 or 5 after theonset of fever (Fig. 1B). The viral load then decreasedin most patients, though it remained detectable ata low level in four of the five patients from whomsamples were obtained on either day 9 or day 10.The detection sensitivity of 79 percent within thefirst three days is better than the reported rate fornasal and throat swabs and is equivalent to that fornasopharyngeal aspirates.3 With the current proto-col, RNA from only 70 µl of plasma was analyzed,and it is likely that centrifugation of a larger volumeof plasma would increase the detection sensitivity.The use of plasma viremia for diagnosing SARS-CoVinfection also has the advantage of not requiringnasopharyngeal aspiration, which is regarded as arisk-prone procedure.5Paul R. Grant, Ph.D.Jeremy A. Garson, M.D., Ph.D.Richard S. Tedder, F.R.C.P., F.R.C.Path.Royal Free and University College Medical SchoolLondon W1T 4JF, United Kingdompaul.grant@ucl.ac.ukFigure 1. Detection of Severe Acute Respiratory Syndrome (SARS)–Associated Coronavirus with a Reverse-Transcriptase Polymerase-Chain-Reaction (PCR) Assay (Panel A) and Changes in the Viral Load (Panel B) in Plasma Samples Obtained up to 10 Days after the Onset of Fever in 26 Patients.Viral infection was considered to have been detected if viral RNA was found by either the nested conventional or real-time PCR method. The number at each time point in Panel B is the number of samples tested.Viral Detection (% of patients)10090807060504030201000 1 2 3 4 5 6 7 8 9 10Days after Onset of FeverAverage Viral Load (copies/ml)16,00014,00012,00010,0008,0006,0004,0002,00000 1 2 3 4 5 6 7 8 9 10Days after Onset of Fever110122 23413855ABDownloaded from www.nejm.org at INST NEUROLOGY on December 18, 2003.Copyright © 2003 Massachusetts Medical Society. All rights reserved.n engl j med 349;25 www.nejm.org december 18, 2003correspondence2469Paul K.S. Chan, M.D.John S. Tam, Ph.D.Joseph J.Y. Sung, M.D., Ph.D.Prince of Wales HospitalShatin, NT, Hong Kong, China1. Ksiazek TG, Erdman D, Goldsmith CS, et al. A novel coronavi-rus associated with severe acute respiratory syndrome. N Engl J Med2003;348:1953-66.2. Lee N, Hui D, Wu A, et al. A major outbreak of severe acute res-piratory syndrome in Hong Kong. N Engl J Med 2003;348:1986-94.3. Poon LL, Wong OK, Luk W, Yuen KY, Peiris JS, Guan Y. Rapid di-agnosis of a coronavirus associated with severe acute respiratorysyndrome (SARS). Clin Chem 2003;49:953-5.4. Drosten C, Gunther S, Preiser W, et al. Identification of a novelcoronavirus in patients with severe acute respiratory syndrome.N Engl J Med 2003;348:1967-76.5. Tsang KW, Mok TY, Wong PC, Ooi GC. Severe acute respiratorysyndrome (SARS) in Hong Kong. Respirology 2003;8:259-65.Estimated Timing of the Last Common Ancestor of the SARS Coronavirusto the editor: Using the viral sequences derivedfrom clinical specimens collected in Hong Kongbetween February and April 2003 from 139 patientswith the severe acute respiratory syndrome (SARS),1we attempted to estimate the timing of the last com-mon ancestor of the coronavirus associated withthe recent outbreaks of SARS. We previously report-ed the results of phylogenetic analysis, using thesame set of spike-gene sequences as that in theSARS-associated coronavirus (SARS-CoV) isolatedduring the early phase of the outbreak (February toApril) in Hong Kong and southern China, togetherwith those available in the public data base.1Assuming that the mutation rate was uniform,we carried out a linear regression analysis that wassimilar to the approach used for the timing of theorigin of human immunodeficiency virus type 1.2The divergence (i.e., branch length), based on thegenetic distance between the isolates and the rootof the phylogram, was plotted as a function of thesampling time (Fig. 1).2,4 The root was defined asthe first diverging point2 of the hypothetical com-mon ancestor of SARS-CoV. The best-fit line wasgenerated by the linear regression analysis extrap-olated back to the past. The divergence of the se-quences from the common ancestor was found tohave a linear relation with time (P<0.001), in a pro-gressive manner. The last appearance of the com-mon ancestor of SARS-CoV was estimated to be onDecember 12, 2002 (95 percent confidence inter-val, September 26, 2002, to January 13, 2003), whenthe value of the divergence was zero. This findingsuggests that the first deviation of the ancestral vi-rus may have occurred in late 2002, close to the timeof the first reports of SARS in southern China.5Although additional sampling of sequences, in-volving a longer time frame, is needed, the currentfindings shed some light on the origin of SARS-CoVand thus may help elucidate its evolution.Figure 1. Timing of the Most Recent Common Ancestor of the Hong Kong SARS-CoV.The divergence of the sequence from the ancestral sequence was plotted against sampling time (GraphPad Prism, version 4.00).3 The red line repre-sents the best-fit line obtained by linear regression analysis, and the blue lines indicate the 95 percent confidence intervals.Divergence (substitutions per site [¬10¡3])2.41.82.02.21.61.41.21.00.80.60.40.20.0July September November January March May2002 2003December12Downloaded from www.nejm.org at INST NEUROLOGY on December 18, 2003.Copyright © 2003 Massachusetts Medical Society. All rights reserved.

Detection of SARS coronavirus in plasma by real-time RT-PCR

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