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Technical Briefs |
Departments of1 Chemical Pathology,2 Paediatrics, 4 Medicine and Therapeutics, and5 Microbiology, The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong SAR;3 Department of Microbiology, The University of Hong Kong, Hong Kong Special Administrative Region
aaddress correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Room 38023, 1/F Clinical Sciences Building, Prince of Wales Hospital, 30--32 Ngan Shing St., Shatin, New Territories, Hong Kong Special Administrative Region, China; e-mail loym{at}cuhk.edu.hk
The recent identification of a novel coronavirus, SARS coronavirus (SARS-CoV), as an etiologic agent for severe acute respiratory syndrome (SARS) has prompted many groups to develop rapid and accurate molecular assays for the detection of this virus (1)(2)(3)(4). To date, most of the assays have focused predominantly on samples taken from nasopharyngeal aspirates, urine, and stools (5)(6). Although detection of SARS-CoV RNA in the plasma of SARS patients has been reported (1), the relatively low sensitivity of the ultracentrifugation-based approach for detecting SARS-CoV RNA in plasma has made this assay impractical. Recently we showed that a one-step real-time quantitative reverse transcription-PCR (RT-PCR) assay for the polymerase region of the SARS-CoV genome could detect viral RNA in 75–78% of nonultracentrifuged serum samples from adult SARS patients during the early stage of disease and that the serum SARS-CoV concentrations on admission were of prognostic significance (7). This finding demonstrates that plasma/serum SARS-CoV quantification may potentially be useful for the early diagnosis of SARS.
Although most existing reports have focused on adult SARS patients, recent reports revealed that the clinical course was less severe in pediatric SARS patients than in adult SARS patients (8)(9). On the whole, the outcomes of pediatric SARS patients have been favorable. In this study, we investigated whether SARS-CoV RNA can be detected in the plasma samples of pediatric patients during different stages of SARS and studied the serial variation in viral loads.
We quantified SARS-CoV RNA by real-time RT-PCR in the plasma of eight pediatric patients admitted to the New Territories East Cluster of Hospital Authority Hospitals in Hong Kong and who satisfied the WHO surveillance case definition for SARS (9). These patients were recruited between March 13, 2003, and May 17, 2003. Informed consent was obtained from the patients or their parents, and ethics approval was obtained from the Institutional Review Board. The serial blood samples used in this study were collected from the patients during sample collection for routine blood tests for monitoring lymphocyte counts and biochemical indices and enzymes. The convalescent sera of these patients were tested for IgG antibody against SARS-CoV with SARS-CoV-infected cells in an indirect immunofluorescence assay (6). All patients were serologically positive for SARS-CoV IgG antibody. As negative controls, blood samples from 15 pediatric patients who suffered from fever and infections other than SARS were collected. The plasma SARS-CoV RNA concentrations in the pediatric patients were compared with the results for adult SARS patients as reported previously (7).
All eight studied patients satisfied the WHO surveillance case definition for SARS (9). Seven of them had been in close contact with infected adults, but one patient had no SARS contact history. All patients had a fever, and the mean duration of the fever was 8 days (range, 4–10 days). During the course of hospitalization, all patients were initially treated with oral ribavirin (40–60 mg/kg daily), which was continued for a mean duration of 10 days (range, 3–14 days). Seven were treated with oral prednisolone starting at a mean of 7 days (range, 6–10 days) after fever onset, and the duration of prednisolone treatment was 14 days.
Blood samples were collected in EDTA-containing tubes and centrifuged at 1600g for 10 min at 4 °C. Plasma was then carefully transferred to plain polypropylene tubes. Viral RNA was extracted from 0.28 mL of plasma with use of a QIAamp viral RNA mini reagent set (Qiagen) as described previously (7).
One-step real-time quantitative RT-PCR was used for SARS-CoV RNA quantification. A RT-PCR system specifically targeting the polymerase gene [orf1ab polyprotein; nt 15327–15398; GenBank accession no. AY278554 (10)] of the SARS-CoV genome was designed as described previously (7). The RT-PCRs were set up in a reaction volume of 25 µL. The primers and fluorescent probe were used at concentrations of 300 and 100 nM, respectively, and 12 µL of extracted plasma RNA was used for amplification. The thermal profile used for the analysis was as follows: the reaction was initiated at 50 °C for 2 min for the included uracil N-glycosylase to act, followed by reverse transcription at 60 °C for 30 min. After a 5-min denaturation at 95 °C, 40 cycles of PCR were carried out with denaturation at 94 °C for 20 s and annealing/extension at 56 °C for 1 min. The sensitivity, linearity, and precision of the assay have been established, as described previously (7). We were able to detect down to 5 copies of the synthetic oligonucleotide in the reaction mixture, which corresponds to 74 copies/mL. SARS-CoV concentrations are expressed as copies/mL of plasma/serum. Because no recovery experiments had been done, the reported concentrations (copies/mL) were minimum estimates. A semilogarithmic plot of different calibrator concentrations against the threshold cycles yielded a correlation coefficient (r) of 0.987. The CV of the copy number of these replicate analyses for this amplification system was 16% at 280 copies/mL (7).
To investigate whether SARS-CoV RNA could be detected in the plasma of pediatric patients, we studied eight patients. The median age of this cohort was 10.3 years (range, 0.3–17.5 years). The earliest available plasma samples were taken from the patients at a mean of 5 days after fever onset (range, 3–7 days), representing a mean of 3 days after admission (range, 1–5 days). SARS-CoV RNA could be detected in seven of the eight (87.5%) pediatric patients (Fig. 1
). The median plasma SARS-CoV RNA concentration was 357 copies/mL. As negative controls, SARS-CoV RNA was not detected in the plasma samples obtained from 15 pediatric patients who suffered from non-SARS-related infections.
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To study the relative usefulness of plasma SARS-CoV measurement at different stages of the disease, we collected serial plasma samples from these eight pediatric SARS patients and subjected them to SARS-CoV quantification. The assay detected SARS-CoV RNA in the plasma of all patients (100%) at a mean of 7 days (range, 6–8 days) after fever onset, representing a mean of 4 days after admission (range, 2–6 days), and the median plasma SARS-CoV RNA concentration was 483 copies/mL. At a mean of 14 days (range, 12–15 days) after fever onset, the detection rate for plasma SARS-CoV dropped to 62.5% (5 of 8), and the median plasma SARS-CoV RNA concentration was 103 copies/mL.
To examine whether the plasma SARS-CoV viral load in pediatric SARS patients is different from that in adult SARS patients, we compared the data from the pediatric patients with the data for 13 adult SARS patients who had been studied in a previous report (7). The adult plasma samples were taken at a mean of 4 days after fever onset (range, 2–6 days) and exactly 7 days after fever onset. The median adult plasma SARS-CoV RNA concentration at a mean of 4 days after fever onset was 125 copies/mL. We observed no significant difference between the plasma SARS-CoV RNA concentration in the pediatric patients and that in the adult SARS patients (Mann–Whitney test, P = 0.076). In addition, we compared the plasma SARS-CoV RNA concentration at day 7 after fever onset. The median adult plasma SARS-CoV RNA concentration at day 7 was 84 copies/mL. Once again, we observed no significant difference between pediatric and adult SARS patients (Mann–Whitney test, P = 0.076). Because the number of patients in this study was limited, further study involving more patients may be necessary to address the difference of viral loads between adult and pediatric SARS patients.
In this study we demonstrated that SARS-CoV RNA is detectable in the plasma of pediatric SARS patients with a detection rate of 87.5–100% within the first week after fever onset and then dropped to 62.5% at a mean of 14 days after fever onset. These data are largely concordant with our previous data on adult SARS patients showing a high detection rate for serum SARS-CoV RNA within the first week of illness (7). Taken together, these data suggest that plasma SARS-CoV measurement is a sensitive method for detecting SARS-CoV infection during the first week of fever onset.
The serial data presented here have demonstrated that SARS-CoV RNA in plasma from the studied patients became undetectable after a mean of 16 days of fever (range, 9–21 days). For patient 7, the undetectable plasma SARS-CoV RNA at days 4 and 10 might represent a fluctuation in the degree of viremia during the course of the illness as a result of intermittent shedding of virions. We did not observe any correlation between the plasma viral load and steroid or ribavirin treatment, and a larger scale study may be necessary to address this important question.
Recent studies have reported that the clinical course is less severe in pediatric SARS patients than in adult SARS patients (8)(9). A logical question would be whether the plasma SARS-CoV viral load in pediatric SARS patients is different from that in adult SARS patients. When we compared the data from pediatric patients with data from adult SARS patients (7), we observed no significant differences in plasma SARS-CoV viral load in samples taken from pediatric and adult SARS patients within the first week of admission and at day 7 after fever onset.
In conclusion, viremia appears to be a consistent feature in both pediatric and adult SARS patients. The relatively high detection of SARS-CoV in plasma during the first week of illness suggests that plasma-based RT-PCR may potentially be useful in the routine diagnostic work-up of patients with suspected or confirmed SARS in both adult and pediatric populations.
Acknowledgments
This work was supported by the Hong Kong Research Grants Council Special Grants for SARS Research (CUHK 4508/03M). We thank Prof. Ambrose King and Prof. Sydney Chung for support during the course of this work.
Footnotes
1 these authors contributed equally to this work 
References
The following articles in journals at HighWire Press have cited this article:
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