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Serial Analysis of the Plasma Concentration of SARS Coronavirus RNA in Pediatric Patients with Severe Acute Respiratory Syndrome

Serial Analysis of the Plasma Concentration of SARS Coronavirus RNA in Pediatric Patients with... Clinical Chemistry 49, No. 12, 2003 2085 potential therapeutic agents and biochemical markers. Ann Clin Biochem still occurred when EDTA plasma was stored at 70 °C 2002;39:551– 6. for 6 months compared with a 61.5% decrease at 20 °C. 5. Yano K, Tsuda E, Washida N, Kobayashi F, Goto M, Harada A, et al. There was no significant difference in OPG concentra- Immunological characterisation of circulating osteoprotegerin/osteoclasto- genesis inhibitory factor: increased serum concentrations in postmeno- tion before and after 6 weeks of storage at 20 or 70 °C pausal women with osteoporosis. J Bone Miner Res 1999;14:518 –27. when samples were collected into either lithium heparin 6. Terpos E, Szydlo R, Apperley JF, Hatjiharissi E, Politou M, Meletis J, et al. or EDTA. Storage of EDTA-plasma samples for up to 6 Soluble receptor activator of nuclear factor B ligand-osteoprotegerin ratio predicts survival in multiple myeloma. Proposal for a novel prognostic index. months at 20 or 70 °C produced a 22.8% decrease in Blood 2003;102:1064 –9. OPG when stored at 20 °C and a 19.7% decrease when 7. Namba N, Manki A, Urakami T, Tanaka H, Seino Y. Serum osteoprotegerin (OPG) levels in children with Juvenile idiopathic arthritis. Bone 2003; stored at 70 °C. 32(Suppl 5):119F. We also investigated the effect of storing separated 8. Browner WS, Lui LY, Cummings SR. Associations of serum osteoprotegerin samples in glass or plastic tubes and observed no signif- levels with diabetes, stroke, bone density, fractures and mortality in elderly women. J Clin Endocrinol Metab 2001;86:631–7. icant effect of tube type. When samples were collected 9. Viereck V, Emons G, Lauck V, Frosch KH, Blaschke S, Grundker C, et al. into nonsiliconized plastic syringes, significant variability Bisphosphonates pamidronate and zoledronic acid stimulate osteoprote- in sRANKL was observed compared with results obtained gerin production by primary human osteoblasts. Biochem Biophys Res Commun 2002;291:680 – 6. when siliconized tubes were used (SARSTEDT- 10. Alvarez L, Peris P, Guanabens N, Vidal S, Ros I, Pons F, et al. Serum MONOVETTE). OPG was not affected by the type of osteoprotegerin and its ligand in Paget’s disease of bone: relationship to disease activity and effect of treatment with bisphosphonates. Arthritis sampling technique used. Rheum 2003;48:824 – 8. The variability in sRANKL concentration under differ- 11. Mackie PS, Fisher JL, Zhou H, Choong PF. Bisphosphonates regulate cell ent storage conditions suggests a possible explanation for growth and gene expression in the UMR 106-01 clonal rat osteosarcoma cell line. Br J Cancer 2001;84:951– 8. previous discordant findings (5, 8 –11 ). The amount of 12. Wuyts W, Van Wesenbeeck L, Morales-Piga A, Ralston S, Hocking L, sRANKL measured by the ELISA used in this study can Vanhoenacker F, et al. Evaluation of the role of RANK and OPG genes in reflect only the free, uncomplexed forms in human plas- Paget’s disease of bone. Bone 2001;28:104 –7. 13. Menaa C, Reddy SV, Kurihara N, Maeda H, Anderson D, Cundy T, et al. ma; therefore, the assays may not accurately reflect the Enhanced RANK ligand expression and responsivity of bone marrow cells in total concentrations of sRANKL and OPG proteins in the Paget’s disease of bone. J Clin Invest 2000;105:1833– 8. samples. Altered concentrations of the proteins could also 14. Cheung J, Mak YT, Papaioannou S, Evans BA, Fogelman I, Hampson G. Interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor B ligand result from fluctuations in the sRANKL:OPG ratio in (RANKL) and osteoprotegerin production by human osteoblastic cells: human plasma, which may contribute to the previous comparison of the effects of 17- oestradiol and raloxifene. J Endocrinol 2003;177:423–33. discordant results for detection of sRANKL:OPG in hu- man plasma. In some circumstances, determination of the DOI: 10.1373/clinchem.2003.023747 expression of RANKL and OPG mRNA extracted from osteoblastic cells may provide a more accurate and precise estimate of changes in the RANKL:OPG ratio in metabolic bone disease than do measurements of plasma concentra- tions (11–14 ). Serial Analysis of the Plasma Concentration of SARS We recommend that samples for measurement of Coronavirus RNA in Pediatric Patients with Severe 1† sRANKL and OPG should be collected into siliconized Acute Respiratory Syndrome, Enders K.O. Ng, Pak- 2† 2 2 syringes or collection tubes and that EDTA is the pre- Cheung Ng, K.L. Ellis Hon, W.T. Frankie Cheng, Emily 2 1 1 ferred anticoagulant. Separation within 1 h and measure- C.W. Hung, K.C. Allen Chan, Rossa W.K. Chiu, Albert M. 2 3 4 5 2 ment of sRANKL and OPG in plasma as soon as possible Li, Leo L.M. Poon, David S. Hui, John S. Tam, Tai-Fai Fok, 1* 1 after collection are also recommended. If required, storage and Y.M. Dennis Lo (Departments of Chemical Pathol- 2 4 5 is best done at 70 °C, but even at this temperature ogy, Paediatrics, Medicine and Therapeutics, and Mi- significant decreases in OPG and sRANKL were observed crobiology, The Chinese University of Hong Kong, Prince at 6 months. of Wales Hospital, Hong Kong SAR; Department of We thank Drs. M.J. Diver and W. Taylor for statistical Microbiology, The University of Hong Kong, Hong Kong and technical assistance. We also thank the Arthritis Special Administrative Region; † these authors contrib- Research Campaign (ARC) for funding of K.A.B. uted equally to this work; * address correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Room 38023, 1/F References 1. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. Clinical Sciences Building, Prince of Wales Hospital, Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation 30--32 Ngan Shing St., Shatin, New Territories, Hong and activation. Cell 1998;93:165–76. Kong Special Administrative Region, China; e-mail 2. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, et [email protected]) al. Osteoclast differentiation factor is a ligand for osteoprotegerin/oste- oclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 1998;95:3597– 602. The recent identification of a novel coronavirus, SARS 3. Nakashima T, Kobayashi Y, Yamasaki S, Kawakami A, Eguchi K, Sasaki H, coronavirus (SARS-CoV), as an etiologic agent for severe et al. Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-B ligand: modulation of the expression by acute respiratory syndrome (SARS) has prompted many osteotrophic factors and cytokines. Biochem Biophys Res Commun 2000; groups to develop rapid and accurate molecular assays 275:768 –75. for the detection of this virus (1– 4). To date, most of the 4. Buckley KA, Fraser WD. Receptor activator for nuclear factor B ligand and osteoprotegerin: regulators of bone physiology and immune responses/ assays have focused predominantly on samples taken from 2086 Technical Briefs nasopharyngeal aspirates, urine, and stools (5, 6 ). Although with use of a QIAamp viral RNA mini reagent set detection of SARS-CoV RNA in the plasma of SARS patients (Qiagen) as described previously (7 ). has been reported (1 ), the relatively low sensitivity of the One-step real-time quantitative RT-PCR was used for ultracentrifugation-based approach for detecting SARS-CoV SARS-CoV RNA quantification. A RT-PCR system specifi- RNA in plasma has made this assay impractical. Recently cally targeting the polymerase gene [orf1ab polyprotein; nt we showed that a one-step real-time quantitative reverse 15327–15398; GenBank accession no. AY278554 (10 )]ofthe transcription-PCR (RT-PCR) assay for the polymerase region SARS-CoV genome was designed as described previously of the SARS-CoV genome could detect viral RNA in 75–78% (7 ). The RT-PCRs were set up in a reaction volume of 25 L. of nonultracentrifuged serum samples from adult SARS The primers and fluorescent probe were used at concentra- patients during the early stage of disease and that the serum tions of 300 and 100 nM, respectively, and 12 L of extracted SARS-CoV concentrations on admission were of prognostic plasma RNA was used for amplification. The thermal profile significance (7 ). This finding demonstrates that plasma/ used for the analysis was as follows: the reaction was serum SARS-CoV quantification may potentially be useful initiated at 50 °C for 2 min for the included uracil N- for the early diagnosis of SARS. glycosylase to act, followed by reverse transcription at 60 °C Although most existing reports have focused on adult for 30 min. After a 5-min denaturation at 95 °C, 40 cycles of SARS patients, recent reports revealed that the clinical PCR were carried out with denaturation at 94 °C for 20 s and course was less severe in pediatric SARS patients than in annealing/extension at 56 °C for 1 min. The sensitivity, adult SARS patients (8, 9 ). On the whole, the outcomes of linearity, and precision of the assay have been established, as pediatric SARS patients have been favorable. In this study, described previously (7 ). We were able to detect down to 5 we investigated whether SARS-CoV RNA can be detected in copies of the synthetic oligonucleotide in the reaction mix- the plasma samples of pediatric patients during different ture, which corresponds to 74 copies/mL. SARS-CoV con- stages of SARS and studied the serial variation in viral loads. centrations are expressed as copies/mL of plasma/serum. We quantified SARS-CoV RNA by real-time RT-PCR in Because no recovery experiments had been done, the re- the plasma of eight pediatric patients admitted to the New ported concentrations (copies/mL) were minimum esti- Territories East Cluster of Hospital Authority Hospitals in mates. A semilogarithmic plot of different calibrator concen- Hong Kong and who satisfied the WHO surveillance case trations against the threshold cycles yielded a correlation definition for SARS (9 ). These patients were recruited coefficient (r) of 0.987. The CV of the copy number of these between March 13, 2003, and May 17, 2003. Informed replicate analyses for this amplification system was 16% at consent was obtained from the patients or their parents, 280 copies/mL (7 ). and ethics approval was obtained from the Institutional To investigate whether SARS-CoV RNA could be de- Review Board. The serial blood samples used in this study tected in the plasma of pediatric patients, we studied were collected from the patients during sample collection eight patients. The median age of this cohort was 10.3 for routine blood tests for monitoring lymphocyte counts years (range, 0.3–17.5 years). The earliest available plasma and biochemical indices and enzymes. The convalescent samples were taken from the patients at a mean of 5 days sera of these patients were tested for IgG antibody against after fever onset (range, 3–7 days), representing a mean of SARS-CoV with SARS-CoV-infected cells in an indirect 3 days after admission (range, 1–5 days). SARS-CoV RNA immunofluorescence assay (6 ). All patients were serolog- could be detected in seven of the eight (87.5%) pediatric ically positive for SARS-CoV IgG antibody. As negative patients (Fig. 1). The median plasma SARS-CoV RNA controls, blood samples from 15 pediatric patients who concentration was 357 copies/mL. As negative controls, suffered from fever and infections other than SARS were SARS-CoV RNA was not detected in the plasma samples collected. The plasma SARS-CoV RNA concentrations in obtained from 15 pediatric patients who suffered from the pediatric patients were compared with the results for non-SARS-related infections. adult SARS patients as reported previously (7 ). To study the relative usefulness of plasma SARS-CoV All eight studied patients satisfied the WHO surveillance measurement at different stages of the disease, we col- case definition for SARS (9 ). Seven of them had been in close lected serial plasma samples from these eight pediatric contact with infected adults, but one patient had no SARS SARS patients and subjected them to SARS-CoV quanti- contact history. All patients had a fever, and the mean fication. The assay detected SARS-CoV RNA in the duration of the fever was 8 days (range, 4 –10 days). During plasma of all patients (100%) at a mean of 7 days (range, the course of hospitalization, all patients were initially 6 – 8 days) after fever onset, representing a mean of 4 days treated with oral ribavirin (40 – 60 mg/kg daily), which was after admission (range, 2– 6 days), and the median plasma continued for a mean duration of 10 days (range, 3–14 days). SARS-CoV RNA concentration was 483 copies/mL. At a Seven were treated with oral prednisolone starting at a mean mean of 14 days (range, 12–15 days) after fever onset, the of 7 days (range, 6 –10 days) after fever onset, and the detection rate for plasma SARS-CoV dropped to 62.5% (5 duration of prednisolone treatment was 14 days. of 8), and the median plasma SARS-CoV RNA concentra- Blood samples were collected in EDTA-containing tion was 103 copies/mL. tubes and centrifuged at 1600g for 10 min at 4 °C. Plasma To examine whether the plasma SARS-CoV viral load in was then carefully transferred to plain polypropylene pediatric SARS patients is different from that in adult tubes. Viral RNA was extracted from 0.28 mL of plasma SARS patients, we compared the data from the pediatric Clinical Chemistry 49, No. 12, 2003 2087 Fig. 1. Serial analysis of plasma SARS-CoV RNA concentrations in pediatric SARS patients. Shown are plots of plasma SARS-CoV RNA concentrations in a common logarithmic scale (copies of SARS-CoV RNA/mL of plasma; y axis) against time after the onset of fever (day 1 refers the day of fever onset; x axis). The horizontal dashed lines represent the detection limit of the assay. 2088 Technical Briefs patients with the data for 13 adult SARS patients who had This work was supported by the Hong Kong Research Grants Council Special Grants for SARS Research (CUHK been studied in a previous report (7 ). The adult plasma 4508/03M). We thank Prof. Ambrose King and Prof. samples were taken at a mean of 4 days after fever onset Sydney Chung for support during the course of this work. (range, 2– 6 days) and exactly 7 days after fever onset. The median adult plasma SARS-CoV RNA concentration at a References mean of 4 days after fever onset was 125 copies/mL. We 1. Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, et al. observed no significant difference between the plasma Identification of a novel coronavirus in patients with severe acute respiratory SARS-CoV RNA concentration in the pediatric patients syndrome. N Engl J Med 2003;348:1967–76. 2. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, et al. A and that in the adult SARS patients (Mann–Whitney test, novel coronavirus associated with severe acute respiratory syndrome. N Engl P  0.076). In addition, we compared the plasma SARS- J Med 2003;348:1953– 66. 3. Fouchier RA, Kuiken T, Schutten M, van Amerongen G, van Doornum GJ, van CoV RNA concentration at day 7 after fever onset. The den Hoogen BG, et al. Aetiology: Koch’s postulates fulfilled for SARS virus. median adult plasma SARS-CoV RNA concentration at Nature 2003;423:240. day 7 was 84 copies/mL. Once again, we observed no 4. Peiris JS, Lai ST, Poon LL, Guan Y, Yam LY, Lim W, et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 2003;361: significant difference between pediatric and adult SARS 1319 –25. patients (Mann–Whitney test, P  0.076). Because the 5. Poon LL, Wong OK, Luk W, Yuen KY, Peiris JS, Guan Y. Rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (SARS). Clin number of patients in this study was limited, further Chem 2003;49:953–5. study involving more patients may be necessary to ad- 6. Peiris JS, Chu CM, Cheng VC, Chan KS, Hung IF, Poon LL, et al. Clinical dress the difference of viral loads between adult and progression and viral load in a community outbreak of coronavirus-associ- ated SARS pneumonia: a prospective study. Lancet 2003;361:1767–72. pediatric SARS patients. 7. Ng EKO, Hui DS, Chan KC, Hung EC, Chiu RW, Lee N, et al. Quantitative In this study we demonstrated that SARS-CoV RNA is analysis and prognostic implication of SARS-coronavirus RNA in the plasma and serum of patients with severe acute respiratory syndrome. Clin Chem detectable in the plasma of pediatric SARS patients with a 2003;49:1976 – 80. detection rate of 87.5–100% within the first week after 8. Chiu WK, Cheung PC, Ng KL, Ip PL, Sugunan VK, Luk DC, et al. Severe acute fever onset and then dropped to 62.5% at a mean of 14 respiratory syndrome in children: experience in a regional hospital in Hong Kong. Pediatr Crit Care Med 2003;4:279 – 83. days after fever onset. These data are largely concordant 9. Hon KL, Leung CW, Cheng WT, Chan PK, Chu WC, Kwan YW, et al. Clinical with our previous data on adult SARS patients showing a presentations and outcome of severe acute respiratory syndrome in chil- dren. Lancet 2003;361:1701–3. high detection rate for serum SARS-CoV RNA within the 10. Tsui SK, Chim SS, Lo YMD, and The Chinese University of Hong Kong first week of illness (7 ). Taken together, these data suggest Molecular SARS Research Group. Coronavirus genomic-sequence variations that plasma SARS-CoV measurement is a sensitive and the epidemiology of the severe acute respiratory syndrome. N Engl J Med 2003;349:187– 8. method for detecting SARS-CoV infection during the first week of fever onset. DOI: 10.1373/clinchem.2003.024588 The serial data presented here have demonstrated that SARS-CoV RNA in plasma from the studied patients be- came undetectable after a mean of 16 days of fever (range, 9 –21 days). For patient 7, the undetectable plasma SARS- Applicability of an Assay for Routine Monitoring of CoV RNA at days 4 and 10 might represent a fluctuation in Highly Variable Concentrations of Olanzapine Based the degree of viremia during the course of the illness as a on HPLC with Mass Spectrometric Detection, Guillermo result of intermittent shedding of virions. We did not ob- 1 1 2 Gervasini, Sonia Vizcaı ´no, Angustias G. Herra ´iz, Julio serve any correlation between the plasma viral load and 1 1* 1 Benı ´tez, and Juan Antonio Carrillo ( Department of steroid or ribavirin treatment, and a larger scale study may Pharmacology and Psychiatry, Extremadura University be necessary to address this important question. School of Medicine, E-06071 Badajoz, Spain; Psychi- Recent studies have reported that the clinical course is atric Hospital Adolfo Dı ´az Ambrona, E-06800 Me ´ rida, less severe in pediatric SARS patients than in adult SARS Spain; * address correspondence to this author at: Depar- patients (8, 9 ). A logical question would be whether the tamento de Farmacologı ´a y Psiquiatrı ´a, Facultad de Me- plasma SARS-CoV viral load in pediatric SARS patients is dicina, Universidad de Extremadura, Avda. de Elvas different from that in adult SARS patients. When we s/n, E-06071 Badajoz, Spain; fax 34-924-271100, e-mail compared the data from pediatric patients with data from [email protected]) adult SARS patients (7 ), we observed no significant differences in plasma SARS-CoV viral load in samples The thienobenzodiazepine derivative olanzapine (OLZ) is taken from pediatric and adult SARS patients within the a commonly used antipsychotic drug that has demon- first week of admission and at day 7 after fever onset. strated efficacy against both positive and negative symp- In conclusion, viremia appears to be a consistent feature toms of schizophrenia (1 ). in both pediatric and adult SARS patients. The relatively OLZ shows variable pharmacokinetics, with induction high detection of SARS-CoV in plasma during the first of the CYP1A2 enzyme by cigarette smoking being one of week of illness suggests that plasma-based RT-PCR may the most important factors contributing to this variability potentially be useful in the routine diagnostic work-up of (2 ). Moreover, because of the potential toxicity of OLZ at patients with suspected or confirmed SARS in both adult relatively low concentrations, monitoring of OLZ has and pediatric populations. been suggested to be necessary (3 ). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Clinical Chemistry Pubmed Central

Serial Analysis of the Plasma Concentration of SARS Coronavirus RNA in Pediatric Patients with Severe Acute Respiratory Syndrome

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Pubmed Central
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© 2003 The American Association for Clinical Chemistry
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0009-9147
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1530-8561
DOI
10.1373/clinchem.2003.024588
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Abstract

Clinical Chemistry 49, No. 12, 2003 2085 potential therapeutic agents and biochemical markers. Ann Clin Biochem still occurred when EDTA plasma was stored at 70 °C 2002;39:551– 6. for 6 months compared with a 61.5% decrease at 20 °C. 5. Yano K, Tsuda E, Washida N, Kobayashi F, Goto M, Harada A, et al. There was no significant difference in OPG concentra- Immunological characterisation of circulating osteoprotegerin/osteoclasto- genesis inhibitory factor: increased serum concentrations in postmeno- tion before and after 6 weeks of storage at 20 or 70 °C pausal women with osteoporosis. J Bone Miner Res 1999;14:518 –27. when samples were collected into either lithium heparin 6. Terpos E, Szydlo R, Apperley JF, Hatjiharissi E, Politou M, Meletis J, et al. or EDTA. Storage of EDTA-plasma samples for up to 6 Soluble receptor activator of nuclear factor B ligand-osteoprotegerin ratio predicts survival in multiple myeloma. Proposal for a novel prognostic index. months at 20 or 70 °C produced a 22.8% decrease in Blood 2003;102:1064 –9. OPG when stored at 20 °C and a 19.7% decrease when 7. Namba N, Manki A, Urakami T, Tanaka H, Seino Y. Serum osteoprotegerin (OPG) levels in children with Juvenile idiopathic arthritis. Bone 2003; stored at 70 °C. 32(Suppl 5):119F. We also investigated the effect of storing separated 8. Browner WS, Lui LY, Cummings SR. Associations of serum osteoprotegerin samples in glass or plastic tubes and observed no signif- levels with diabetes, stroke, bone density, fractures and mortality in elderly women. J Clin Endocrinol Metab 2001;86:631–7. icant effect of tube type. When samples were collected 9. Viereck V, Emons G, Lauck V, Frosch KH, Blaschke S, Grundker C, et al. into nonsiliconized plastic syringes, significant variability Bisphosphonates pamidronate and zoledronic acid stimulate osteoprote- in sRANKL was observed compared with results obtained gerin production by primary human osteoblasts. Biochem Biophys Res Commun 2002;291:680 – 6. when siliconized tubes were used (SARSTEDT- 10. Alvarez L, Peris P, Guanabens N, Vidal S, Ros I, Pons F, et al. Serum MONOVETTE). OPG was not affected by the type of osteoprotegerin and its ligand in Paget’s disease of bone: relationship to disease activity and effect of treatment with bisphosphonates. Arthritis sampling technique used. Rheum 2003;48:824 – 8. The variability in sRANKL concentration under differ- 11. Mackie PS, Fisher JL, Zhou H, Choong PF. Bisphosphonates regulate cell ent storage conditions suggests a possible explanation for growth and gene expression in the UMR 106-01 clonal rat osteosarcoma cell line. Br J Cancer 2001;84:951– 8. previous discordant findings (5, 8 –11 ). The amount of 12. Wuyts W, Van Wesenbeeck L, Morales-Piga A, Ralston S, Hocking L, sRANKL measured by the ELISA used in this study can Vanhoenacker F, et al. Evaluation of the role of RANK and OPG genes in reflect only the free, uncomplexed forms in human plas- Paget’s disease of bone. Bone 2001;28:104 –7. 13. Menaa C, Reddy SV, Kurihara N, Maeda H, Anderson D, Cundy T, et al. ma; therefore, the assays may not accurately reflect the Enhanced RANK ligand expression and responsivity of bone marrow cells in total concentrations of sRANKL and OPG proteins in the Paget’s disease of bone. J Clin Invest 2000;105:1833– 8. samples. Altered concentrations of the proteins could also 14. Cheung J, Mak YT, Papaioannou S, Evans BA, Fogelman I, Hampson G. Interleukin-6 (IL-6), IL-1, receptor activator of nuclear factor B ligand result from fluctuations in the sRANKL:OPG ratio in (RANKL) and osteoprotegerin production by human osteoblastic cells: human plasma, which may contribute to the previous comparison of the effects of 17- oestradiol and raloxifene. J Endocrinol 2003;177:423–33. discordant results for detection of sRANKL:OPG in hu- man plasma. In some circumstances, determination of the DOI: 10.1373/clinchem.2003.023747 expression of RANKL and OPG mRNA extracted from osteoblastic cells may provide a more accurate and precise estimate of changes in the RANKL:OPG ratio in metabolic bone disease than do measurements of plasma concentra- tions (11–14 ). Serial Analysis of the Plasma Concentration of SARS We recommend that samples for measurement of Coronavirus RNA in Pediatric Patients with Severe 1† sRANKL and OPG should be collected into siliconized Acute Respiratory Syndrome, Enders K.O. Ng, Pak- 2† 2 2 syringes or collection tubes and that EDTA is the pre- Cheung Ng, K.L. Ellis Hon, W.T. Frankie Cheng, Emily 2 1 1 ferred anticoagulant. Separation within 1 h and measure- C.W. Hung, K.C. Allen Chan, Rossa W.K. Chiu, Albert M. 2 3 4 5 2 ment of sRANKL and OPG in plasma as soon as possible Li, Leo L.M. Poon, David S. Hui, John S. Tam, Tai-Fai Fok, 1* 1 after collection are also recommended. If required, storage and Y.M. Dennis Lo (Departments of Chemical Pathol- 2 4 5 is best done at 70 °C, but even at this temperature ogy, Paediatrics, Medicine and Therapeutics, and Mi- significant decreases in OPG and sRANKL were observed crobiology, The Chinese University of Hong Kong, Prince at 6 months. of Wales Hospital, Hong Kong SAR; Department of We thank Drs. M.J. Diver and W. Taylor for statistical Microbiology, The University of Hong Kong, Hong Kong and technical assistance. We also thank the Arthritis Special Administrative Region; † these authors contrib- Research Campaign (ARC) for funding of K.A.B. uted equally to this work; * address correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Room 38023, 1/F References 1. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al. Clinical Sciences Building, Prince of Wales Hospital, Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation 30--32 Ngan Shing St., Shatin, New Territories, Hong and activation. Cell 1998;93:165–76. Kong Special Administrative Region, China; e-mail 2. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, Mochizuki S, et [email protected]) al. Osteoclast differentiation factor is a ligand for osteoprotegerin/oste- oclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc Natl Acad Sci U S A 1998;95:3597– 602. The recent identification of a novel coronavirus, SARS 3. Nakashima T, Kobayashi Y, Yamasaki S, Kawakami A, Eguchi K, Sasaki H, coronavirus (SARS-CoV), as an etiologic agent for severe et al. Protein expression and functional difference of membrane-bound and soluble receptor activator of NF-B ligand: modulation of the expression by acute respiratory syndrome (SARS) has prompted many osteotrophic factors and cytokines. Biochem Biophys Res Commun 2000; groups to develop rapid and accurate molecular assays 275:768 –75. for the detection of this virus (1– 4). To date, most of the 4. Buckley KA, Fraser WD. Receptor activator for nuclear factor B ligand and osteoprotegerin: regulators of bone physiology and immune responses/ assays have focused predominantly on samples taken from 2086 Technical Briefs nasopharyngeal aspirates, urine, and stools (5, 6 ). Although with use of a QIAamp viral RNA mini reagent set detection of SARS-CoV RNA in the plasma of SARS patients (Qiagen) as described previously (7 ). has been reported (1 ), the relatively low sensitivity of the One-step real-time quantitative RT-PCR was used for ultracentrifugation-based approach for detecting SARS-CoV SARS-CoV RNA quantification. A RT-PCR system specifi- RNA in plasma has made this assay impractical. Recently cally targeting the polymerase gene [orf1ab polyprotein; nt we showed that a one-step real-time quantitative reverse 15327–15398; GenBank accession no. AY278554 (10 )]ofthe transcription-PCR (RT-PCR) assay for the polymerase region SARS-CoV genome was designed as described previously of the SARS-CoV genome could detect viral RNA in 75–78% (7 ). The RT-PCRs were set up in a reaction volume of 25 L. of nonultracentrifuged serum samples from adult SARS The primers and fluorescent probe were used at concentra- patients during the early stage of disease and that the serum tions of 300 and 100 nM, respectively, and 12 L of extracted SARS-CoV concentrations on admission were of prognostic plasma RNA was used for amplification. The thermal profile significance (7 ). This finding demonstrates that plasma/ used for the analysis was as follows: the reaction was serum SARS-CoV quantification may potentially be useful initiated at 50 °C for 2 min for the included uracil N- for the early diagnosis of SARS. glycosylase to act, followed by reverse transcription at 60 °C Although most existing reports have focused on adult for 30 min. After a 5-min denaturation at 95 °C, 40 cycles of SARS patients, recent reports revealed that the clinical PCR were carried out with denaturation at 94 °C for 20 s and course was less severe in pediatric SARS patients than in annealing/extension at 56 °C for 1 min. The sensitivity, adult SARS patients (8, 9 ). On the whole, the outcomes of linearity, and precision of the assay have been established, as pediatric SARS patients have been favorable. In this study, described previously (7 ). We were able to detect down to 5 we investigated whether SARS-CoV RNA can be detected in copies of the synthetic oligonucleotide in the reaction mix- the plasma samples of pediatric patients during different ture, which corresponds to 74 copies/mL. SARS-CoV con- stages of SARS and studied the serial variation in viral loads. centrations are expressed as copies/mL of plasma/serum. We quantified SARS-CoV RNA by real-time RT-PCR in Because no recovery experiments had been done, the re- the plasma of eight pediatric patients admitted to the New ported concentrations (copies/mL) were minimum esti- Territories East Cluster of Hospital Authority Hospitals in mates. A semilogarithmic plot of different calibrator concen- Hong Kong and who satisfied the WHO surveillance case trations against the threshold cycles yielded a correlation definition for SARS (9 ). These patients were recruited coefficient (r) of 0.987. The CV of the copy number of these between March 13, 2003, and May 17, 2003. Informed replicate analyses for this amplification system was 16% at consent was obtained from the patients or their parents, 280 copies/mL (7 ). and ethics approval was obtained from the Institutional To investigate whether SARS-CoV RNA could be de- Review Board. The serial blood samples used in this study tected in the plasma of pediatric patients, we studied were collected from the patients during sample collection eight patients. The median age of this cohort was 10.3 for routine blood tests for monitoring lymphocyte counts years (range, 0.3–17.5 years). The earliest available plasma and biochemical indices and enzymes. The convalescent samples were taken from the patients at a mean of 5 days sera of these patients were tested for IgG antibody against after fever onset (range, 3–7 days), representing a mean of SARS-CoV with SARS-CoV-infected cells in an indirect 3 days after admission (range, 1–5 days). SARS-CoV RNA immunofluorescence assay (6 ). All patients were serolog- could be detected in seven of the eight (87.5%) pediatric ically positive for SARS-CoV IgG antibody. As negative patients (Fig. 1). The median plasma SARS-CoV RNA controls, blood samples from 15 pediatric patients who concentration was 357 copies/mL. As negative controls, suffered from fever and infections other than SARS were SARS-CoV RNA was not detected in the plasma samples collected. The plasma SARS-CoV RNA concentrations in obtained from 15 pediatric patients who suffered from the pediatric patients were compared with the results for non-SARS-related infections. adult SARS patients as reported previously (7 ). To study the relative usefulness of plasma SARS-CoV All eight studied patients satisfied the WHO surveillance measurement at different stages of the disease, we col- case definition for SARS (9 ). Seven of them had been in close lected serial plasma samples from these eight pediatric contact with infected adults, but one patient had no SARS SARS patients and subjected them to SARS-CoV quanti- contact history. All patients had a fever, and the mean fication. The assay detected SARS-CoV RNA in the duration of the fever was 8 days (range, 4 –10 days). During plasma of all patients (100%) at a mean of 7 days (range, the course of hospitalization, all patients were initially 6 – 8 days) after fever onset, representing a mean of 4 days treated with oral ribavirin (40 – 60 mg/kg daily), which was after admission (range, 2– 6 days), and the median plasma continued for a mean duration of 10 days (range, 3–14 days). SARS-CoV RNA concentration was 483 copies/mL. At a Seven were treated with oral prednisolone starting at a mean mean of 14 days (range, 12–15 days) after fever onset, the of 7 days (range, 6 –10 days) after fever onset, and the detection rate for plasma SARS-CoV dropped to 62.5% (5 duration of prednisolone treatment was 14 days. of 8), and the median plasma SARS-CoV RNA concentra- Blood samples were collected in EDTA-containing tion was 103 copies/mL. tubes and centrifuged at 1600g for 10 min at 4 °C. Plasma To examine whether the plasma SARS-CoV viral load in was then carefully transferred to plain polypropylene pediatric SARS patients is different from that in adult tubes. Viral RNA was extracted from 0.28 mL of plasma SARS patients, we compared the data from the pediatric Clinical Chemistry 49, No. 12, 2003 2087 Fig. 1. Serial analysis of plasma SARS-CoV RNA concentrations in pediatric SARS patients. Shown are plots of plasma SARS-CoV RNA concentrations in a common logarithmic scale (copies of SARS-CoV RNA/mL of plasma; y axis) against time after the onset of fever (day 1 refers the day of fever onset; x axis). The horizontal dashed lines represent the detection limit of the assay. 2088 Technical Briefs patients with the data for 13 adult SARS patients who had This work was supported by the Hong Kong Research Grants Council Special Grants for SARS Research (CUHK been studied in a previous report (7 ). The adult plasma 4508/03M). We thank Prof. Ambrose King and Prof. samples were taken at a mean of 4 days after fever onset Sydney Chung for support during the course of this work. (range, 2– 6 days) and exactly 7 days after fever onset. The median adult plasma SARS-CoV RNA concentration at a References mean of 4 days after fever onset was 125 copies/mL. We 1. Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, et al. observed no significant difference between the plasma Identification of a novel coronavirus in patients with severe acute respiratory SARS-CoV RNA concentration in the pediatric patients syndrome. N Engl J Med 2003;348:1967–76. 2. Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, et al. A and that in the adult SARS patients (Mann–Whitney test, novel coronavirus associated with severe acute respiratory syndrome. N Engl P  0.076). In addition, we compared the plasma SARS- J Med 2003;348:1953– 66. 3. Fouchier RA, Kuiken T, Schutten M, van Amerongen G, van Doornum GJ, van CoV RNA concentration at day 7 after fever onset. The den Hoogen BG, et al. Aetiology: Koch’s postulates fulfilled for SARS virus. median adult plasma SARS-CoV RNA concentration at Nature 2003;423:240. day 7 was 84 copies/mL. Once again, we observed no 4. Peiris JS, Lai ST, Poon LL, Guan Y, Yam LY, Lim W, et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 2003;361: significant difference between pediatric and adult SARS 1319 –25. patients (Mann–Whitney test, P  0.076). Because the 5. Poon LL, Wong OK, Luk W, Yuen KY, Peiris JS, Guan Y. Rapid diagnosis of a coronavirus associated with severe acute respiratory syndrome (SARS). Clin number of patients in this study was limited, further Chem 2003;49:953–5. study involving more patients may be necessary to ad- 6. Peiris JS, Chu CM, Cheng VC, Chan KS, Hung IF, Poon LL, et al. Clinical dress the difference of viral loads between adult and progression and viral load in a community outbreak of coronavirus-associ- ated SARS pneumonia: a prospective study. Lancet 2003;361:1767–72. pediatric SARS patients. 7. Ng EKO, Hui DS, Chan KC, Hung EC, Chiu RW, Lee N, et al. Quantitative In this study we demonstrated that SARS-CoV RNA is analysis and prognostic implication of SARS-coronavirus RNA in the plasma and serum of patients with severe acute respiratory syndrome. Clin Chem detectable in the plasma of pediatric SARS patients with a 2003;49:1976 – 80. detection rate of 87.5–100% within the first week after 8. Chiu WK, Cheung PC, Ng KL, Ip PL, Sugunan VK, Luk DC, et al. Severe acute fever onset and then dropped to 62.5% at a mean of 14 respiratory syndrome in children: experience in a regional hospital in Hong Kong. Pediatr Crit Care Med 2003;4:279 – 83. days after fever onset. These data are largely concordant 9. Hon KL, Leung CW, Cheng WT, Chan PK, Chu WC, Kwan YW, et al. Clinical with our previous data on adult SARS patients showing a presentations and outcome of severe acute respiratory syndrome in chil- dren. Lancet 2003;361:1701–3. high detection rate for serum SARS-CoV RNA within the 10. Tsui SK, Chim SS, Lo YMD, and The Chinese University of Hong Kong first week of illness (7 ). Taken together, these data suggest Molecular SARS Research Group. Coronavirus genomic-sequence variations that plasma SARS-CoV measurement is a sensitive and the epidemiology of the severe acute respiratory syndrome. N Engl J Med 2003;349:187– 8. method for detecting SARS-CoV infection during the first week of fever onset. DOI: 10.1373/clinchem.2003.024588 The serial data presented here have demonstrated that SARS-CoV RNA in plasma from the studied patients be- came undetectable after a mean of 16 days of fever (range, 9 –21 days). For patient 7, the undetectable plasma SARS- Applicability of an Assay for Routine Monitoring of CoV RNA at days 4 and 10 might represent a fluctuation in Highly Variable Concentrations of Olanzapine Based the degree of viremia during the course of the illness as a on HPLC with Mass Spectrometric Detection, Guillermo result of intermittent shedding of virions. We did not ob- 1 1 2 Gervasini, Sonia Vizcaı ´no, Angustias G. Herra ´iz, Julio serve any correlation between the plasma viral load and 1 1* 1 Benı ´tez, and Juan Antonio Carrillo ( Department of steroid or ribavirin treatment, and a larger scale study may Pharmacology and Psychiatry, Extremadura University be necessary to address this important question. School of Medicine, E-06071 Badajoz, Spain; Psychi- Recent studies have reported that the clinical course is atric Hospital Adolfo Dı ´az Ambrona, E-06800 Me ´ rida, less severe in pediatric SARS patients than in adult SARS Spain; * address correspondence to this author at: Depar- patients (8, 9 ). A logical question would be whether the tamento de Farmacologı ´a y Psiquiatrı ´a, Facultad de Me- plasma SARS-CoV viral load in pediatric SARS patients is dicina, Universidad de Extremadura, Avda. de Elvas different from that in adult SARS patients. When we s/n, E-06071 Badajoz, Spain; fax 34-924-271100, e-mail compared the data from pediatric patients with data from [email protected]) adult SARS patients (7 ), we observed no significant differences in plasma SARS-CoV viral load in samples The thienobenzodiazepine derivative olanzapine (OLZ) is taken from pediatric and adult SARS patients within the a commonly used antipsychotic drug that has demon- first week of admission and at day 7 after fever onset. strated efficacy against both positive and negative symp- In conclusion, viremia appears to be a consistent feature toms of schizophrenia (1 ). in both pediatric and adult SARS patients. The relatively OLZ shows variable pharmacokinetics, with induction high detection of SARS-CoV in plasma during the first of the CYP1A2 enzyme by cigarette smoking being one of week of illness suggests that plasma-based RT-PCR may the most important factors contributing to this variability potentially be useful in the routine diagnostic work-up of (2 ). Moreover, because of the potential toxicity of OLZ at patients with suspected or confirmed SARS in both adult relatively low concentrations, monitoring of OLZ has and pediatric populations. been suggested to be necessary (3 ).

Journal

Clinical Chemistry – Pubmed Central

Published: Dec 1, 2003

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