J S M Peiris, S T Lai, L L M Poon, Y Guan, L Y C Yam, W Lim, J Nicholls, W K S Yee, W W Yan, M T Cheung, V C C Cheng, K H Chan, D N C Tsang, R W H Yung, T K Ng, K Y Yuen, and members of the SARS study group*

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*Members listed at the end of paper

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Department of Microbiology and Pathology, Queen Mary Hospital, University of Hong Kong, Hong Kong (Prof J S M Peiris DPhil, L L M Poon DPhil, Y Guan PhD, J Nicholls FRCPA, V C C Cheng MRCP, K H Chan PhD, Prof K Y Yuen FRCPath); Department of Medicine, Intensive Care and Pathology, Princess Margaret Hospital, Hong Kong (S T Lai FRCP, W W Yan FRCP, T K Ng FRCPath); Government Virus Unit, Department of Health, Hong Kong (W Lim FRCPath); Department of Medicine and Pathology, Pamela Youde Nethersole Eastern Hospital, Hong Kong (L Y C Yam FRCP, M T Cheung MRCP, R W H Yung FRCPath); Department of Medicine, Kwong Wah Hospital, Hong Kong (W K S Yee MRCP); and Department of Pathology, Queen Elizabeth Hospital, Hong Kong Special Administrative Region, China (D N C Tsang FRCPath)

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Correspondence to: Prof J S M Peiris, Department of Microbiology, University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Special Administrative Region, China (e-mail:malik@hkucc.hku.hk)Summary
Introduction
Methods
Results
Discussion
References

All 50 patients with SARS were ethnic Chinese. They represented five different epidemiologically linked clusters and sporadic cases fitting the case definition. They were admitted to hospital at a mean of 5 days (SD 2·3) after the onset of symptoms. The median age was 42 years (range 23-74) and the female-to-male ratio was 1 to 1·3. Among the patients, 14 (28%) were health-care workers and five (10%) had a history of a visit to a hospital in which there was a major outbreak of SARS, 13 (26%) were household contacts and 12 (24%) had social contacts with patients who had SARS; four (8%) had recently travelled to mainland China.The presenting complaint in most patients was feverishness or shortness of breath. Cough and myalgia were present in more than half the patients (table 1). Upper-respiratory-tract symptoms such as rhinorrhoea (n=12, 24%) and sore throat (n=10, 20%) were present in a few patients. Watery diarrhoea (n=5, 10%) and anorexia (n=5, 10%) were also reported. At initial examination, auscultatory findings such as crepitations and decreased air entry were present in only 19 (38%) patients. Dry cough was reported by 31 (62%) patients. All had radiological evidence of consolidation at the time of admission involving one zone in 36, two zones in 13, and three zones in one.

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Clinical symptoms*	Number (%)
Fever	50 (100)
Chill or rigors	37 (74)
Cough	31 (62)
Myalgia	27 (54)
Malaise	25 (50)
Running nose	12 (24)
Sore throat	10 (20)
Shortness of breath	10 (20)
Anorexia	10 (20)
Diarrhoea	5 (10)
Headache	10 (20)
Dizziness	6 (12)
*Truncal maculopapular rash was noted in one patient.
Table 1: Symptoms of 50 patients with SARS at presentation

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Despite high fever, 49 (98%) patients had no evidence of a leucocytosis. In peripheral blood tests lymphopenia was present in 68%, leucopenia in 26%, thrombocytopenia in 40%, and anaemia in 18% (table 2). Alanine aminotransferase (45-350 U/L) and creatinine kinase (141-1379 U/L) were raised in 34% and 26%, respectively.

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Laboratory variables		Mean (range)	Number (%) of abnormal	Normal range
Haemoglobin		12·9 (8·9-15·9)	··	11·5-16·5 g/dL
	Anaemia	··	9 (18%)	··
White-cell count		5·17 (1·1-11·4)	··	4-11x109/L
	Leucopenia	··	13 (26%)	··
Lymphocyte count		0·78 (0·3-1·5)	··	1·5-4·0x109/L
	Severe lymphopenia (<1·0x109 /L)	··	34 (68%)	··
Platelet count		174 (88-351)	··	150-400x109/L
	Thrombocytopenia	··	20 (40%)	··
Alanine aminotransferase		63 (11-350)	··	6-53 U/L
	Raised alanine aminotransferase	··	17 (34%)	··
Albumin		37 (26-50)	··	42-54 g/L
	Low albumin	··	34 (68%)	··
Globulin		33 (21-42)	··	24-36 g/L
	Raised globulin	··	10 (20%)	··
Creatinine kinase		244 (31-1379)	··	34-138 U/L
	Raised creatinine kinase	··	13 (26%)	··
Table 2: Initial laboratory findings of 50 patients with SARS

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Routine microbiological investigation for known viruses and bacteria by culture, antigen detection, and PCR was negative in most cases. Blood culture was positive for Escherichia coli in one man aged 74 years admitted to intensive care. The finding was attributed to a hospital-acquired urinary-tract infection. Klebsiella pneumoniae and Haemophilus influenzae were isolated from the sputum samples of two other patients on admission.Oral levofloxacin 500 mg every 24 h was given to nine patients, and amoxicillin-clavulanate given intravenously 1·2 g at 8 h intervals or orally 375 mg three times daily, and intravenous or oral clarithromycin 500 mg every 12 h were given to another 40 patients. Four patients received oral oseltamivir 75 mg twice daily. In one patient, intravenous ceftriaxone 2 g every 24 h, oral azithromycin 500 mg every 24 h, and oral amantadine 100 mg twice daily were given for empirical coverage of typical and atypical pneumonia.At the time of writing, 19 patients had progressed to severe disease with oxygen desaturation requiring intensive care and ventilatory support for a mean of 6·4 days. The mean time between onset of symptoms and worsening was 8·3 days. Intravenous ribavirin 8 mg/kg every 8 h for 7-10 days and steroid (intravenous hydrocortisone 100 mg every 6 h, or hydrocortisone 200 mg every 8 h, or methylprednisolone 1-3 mg/kg every 24 h for two to three doses and tailed off over 2-3 weeks) was given in 49 patients at a mean of 6·7 days after onset of symptoms. Of the six patients given ribavirin and steroids before intubation and ventilation in intensive care, two had a consistent response in terms of resolution of fever, decreased respiratory support, and later radiological resolution, whereas the other four had fluctuating fever and static requirement in respiratory support.The risk factors associated with severe complicated disease requiring intensive care and ventilatory support were older age, severe lymphopenia, impaired alanine aminotransferase, and delayed starting of ribavirin and steroid (table 3). All the complicated cases were treated with ribavirin and steroids after admission to the intensive-care unit, whereas all the uncomplicated cases were started on ribavirin and steroids in the general ward. As expected, 31 uncomplicated cases recovered or improved, whereas eight patients with complicated disease worsened, with one death at the time of writing. All 50 patients had been monitored for a mean of 12 days (SD 6·1) at the time of writing.

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		Complicated case (n=19)	Uncomplicated case (n=31)	p
Mean (SD) age		49·5 (12·7)	39·0 (10·7)	0·005
Male/female ratio		8/11	14/17
Underlying illness		5*	1†	0·05
Method of contact
	Travel to China	1	3
	Health-care worker	5	9
	Hospital visit	1	4
	Household contact	8	5	0·09
	Social contact	4	10
Mean (SD) duration of symptoms to admission (days)		5·2 (2·0)	4·7 (2·5)
Mean (SD) admission temperature (°C)		38·8 (0·9)	38·7 (0·8)
Mean (SD) initial total peripheral WBC count (x109/L)		5·1 (2·4)	5·2 (1·8)
Mean (SD) initial lymphocyte count (x109/L)		0·66 (0·3)	0·85 (0·3)	0·04
Presence of thrombocytopenia (<150x109/L)		8	12
Impaired liver-function test		11	6	0·01
Chest radiographic changes (number of zones affected)		1·4	1·2
Mean (SD) day of worsening from onset of symptoms‡		8·3 (2·6)	Not applicable
Number of patients who received ribavirin and steroids		18	31
Mean (SD) day of start of ribavirin and steroids from onset of symptoms		7·7 (2·9)	5·7 (2·6)	0·03
Start of ribavirin and steroids after worsening		12	0	0·0001
Response to ribavirin and steroids§		11	28	0·02
Outcome
	Improved or recovered	10	31	0·0001
	Not improving	8	0	0·0004
	Died	1	0
WBC=white-blood cell. *Two patients had diabetes mellitus, one had hypertrophic obstructive cardiomyopathy, one had chronic active hepatitis B, and one had brain tumour. †One patient had essential hypertension. ‡Desaturation requiring intensive-care support. §Response defined as resolution of fever within 48 h, decreased ventilatory support, or radiological improvement.
Table 3: Risk factors associated with severe disease requiring intensive care and ventilatory support

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Two virus isolates, identified as a coronavirus, were isolated from two patients. One was from an open lung biopsy sample from a male Hong Kong Chinese resident aged 53 years and the other from a nasopharyngeal aspirate of a woman aged 42 years with good previous health. The man had a history of 10 h social contact with a Chinese visitor coming from Guangzhou, mainland China, who later died from SARS. 2 days after exposure, this patient presented with fever, malaise, myalgia, and headache. Crepitations were present over the right lower zone and there was a corresponding alevolar shadow on the chest radiograph. Haematological investigation revealed lymphopenia of 0·7x10⁹/L with normal total white-cell and platelet count. Alanine aminotransferase (41 U/L) and creatinine phosphokinase (405 U/L) were impaired. Despite a combination of oral azithromycin, amantadine, and intravenous ceftriaxone, there was increasing bilateral pulmonary infiltrates and progressive oxygen desaturation. Therefore, an open lung biopsy was done 9 days after admission. Histopathological examination showed a mild interstitial inflammation with scattered alveolar pneumocytes showing cytomegaly, granular amphophilic cytoplasm, and enlarged nuclei with prominent nucleoli. No cells showed inclusions typical of herpes virus or adenovirus infection. He required ventilation and intensive care after the surgical procedure. Empirical intravenous ribavirin and hydrocortisone were given. He died 20 days after admission. Coronavirus RNA was detected in his nasopharyngeal aspirate, lung biopsy samples, and post-mortem lung samples. He had a significant rise in antibody titre (from 1/200 to 1/1600) to his own coronavirus isolate.The female patient from whom a coronavirus was isolated had a history of good health. She had recently travelled to Guangzhou for 2 days. She presented with fever and diarrhoea 5 days after return to Hong Kong. Physical examination showed crepitation over the right lower zone, which had a corresponding alveolar shadow on chest radiograph. Investigation revealed leucopenia (2·7x10⁹/L), lymphopenia (0·6x10⁹/L), and thrombocytopenia (104x10⁹/L). Despite empirical antimicrobial coverage with amoxicillin-clavulanate, clarithromycin, and oseltamivir, she worsened 5 days after admission and required mechanical ventilation and intensive care for 5 days. She gradually improved without treatment by ribavirin or steroids. Her nasopharyngeal aspirate was positive on RT-PCR for coronavirus and she seroconverted from titre less than one per 50 to one per 1600 to the coronavirus isolate.Viruses were isolated on fetal rhesus kidney cells from the lung biopsy and nasopharyngeal aspirate, respectively, of these two patients. The initial cytopathic effect noted was the appearance of rounded refractile cells appearing 2-4 days after inoculation. The cytopathic effect did not progress in the initial culture tubes but on subsequent passage, and appeared in 24 h. The two virus isolates did not react with the routine panel of reagents used to identify virus isolates, including those to influenza A, B, parainfluenza types 1, 2, and 3, adenovirus, and respiratory syncytial virus (DAKO, Glostrup, Denmark). They also did not react in RT-PCR assays for influenza A and human metapneumovirus, or in PCR assays for mycoplasma. The virus was ether sensitive, which shows that it was an enveloped virus. Electron microscopy of negative stained (3% potassium phospho-tungstate, pH 7·0) ultracentrifuged cell-culture extracts showed the presence of pleomorphic enveloped virus particles of around 80-90 nm (range 70-130 nm) in diameter with surface morphology compatible with a coronavirus (figure 1). Thin-section electron microscopy of infected cells revealed virus particles of 55-90 nm diameter within smooth walled vesicles in the cytoplasm (figure 2, B). Virus particles were also seen at the cell surface. The overall findings were compatible with coronavirus infection in the cells.

Figure 1: Electron microscopy of ultracentrifuged deposit of cell-culture-grown human pneumonia-associated coronavirusNegatively stained with 3% potassium phospho-tungstate, pH 7·0.

Figure 2: Thin-section electron micrograph of lung biopsy sample from patient with SARS (A) and of human pneumonia-associated coronavirus infected cells (B)

A thin-section electron micrograph of the lung biopsy sample from the 53-year-old male contained 60-90 nm viral particles in the cytoplasm of desquamated cells. These viral particles were similar in size and morphology to those observed in the cell cultured virus isolate from both patients (figure 2, A).The RT-PCR products generated in a random primer RT-PCR assay were analysed, and unique bands found in the virus-infected samples were cloned and sequenced. Of 30 clones examined, one containing 646 bp of unknown origin was identified. Sequence analysis of this DNA fragment suggested this sequence had a weak homology to viruses of the family of Coronaviridae. Deducted aminoacid sequence (215 aminoacids) from this unknown sequence, however, had the highest homology (57%) to the RNA polymerase of bovine coronavirus and murine hepatitis virus, confirming that this virus belongs to the family of Coronaviridae. Phylogenetic analysis of the protein sequences showed that this virus, although most closely related to the group II coronaviruses, was a distinct virus (figure 3).

Figure 3: Phylogenetic analysis of the partial protein sequence (215 aminoacids) of the coronavirus (SARS)GenBank accession number AY268070. Tree is constructed by neighbour-jointing method. Horizontal-line distance represents number of sites at which the two sequences compared are different. Bootstrap values deducted from 500 replicates.

Based on the 646 bp sequence of the isolate, specific primers for detecting the new virus were designed for RT-PCR detection of this human pneumonia-associated coronavirus genome in clinical samples. Of the 44 nasopharyngeal samples available from the 50 SARS patients, 22 had evidence of human pneumonia-associated coronavirus RNA. Viral RNA was detectable in ten of 18 faecal samples tested. The specificity of the RT-PCR reaction was confirmed by sequencing selected positive RT-PCR-amplified products. None of 40 nasophararyngeal and faecal samples from patients with unrelated diseases were reactive on RT-PCR.In 35 of the 50 most recent serum samples from patients with SARS there was evidence of antibody to the coronavirus. Of 32 patients from whom paired acute and convalescent sera were available, all had seroconverted or had more than a four-fold increase in antibody titre to the virus. Five other pairs of sera from additional SARS patients from clusters outside this study group were also tested to provide a wider sampling of SARS patients in the community and all of them seroconverted. None of 80 sera from patients with respiratory or other diseases and none of 200 blood donors had detectable antibody.If seropositivity to human pneumonia-associated coronavirus in one serum sample or viral RNA detection in the nasopharyngeal aspirates or stools is deemed evidence of infection with the coronavirus, 45 of the 50 patients have evidence of infection. Of the five patients with no virological evidence of coronavirus infection, only one had a serum sample tested more than 14 days after onset of clinical disease.