[SARS-associated coronavirus gene fragments were detected from a suspected pediatric SARS patient]

Ru-nan Zhu, Yuan Qian, Jie Deng, Lin-qing Zhao, Fang Wang, Li Cao, Tian-you Wang, Da-kun Chen, Qi Zhang
Zhonghua Er Ke za Zhi. Chinese Journal of Pediatrics 2003, 41 (9): 641-4

UNLABELLED: A Special "Fever and Cough" Clinic was set up at the Children's Hospital Affiliated to Capital Institute of Pediatrics for children with symptoms of fever and cough in late April when the severe acute respiratory syndrome (SARS) epidemic was at its peak in Beijing to separate the children with fever from others during their visit to the Outpatient Department.

OBJECTIVE: For patients with fever, normal or low count of white blood cell and with suspected pneumonia suggested by X-ray, it was urgent to determine the etiological agents of the diseases before they were admitted to the hospital.

METHODS: Throat swabs or nasopharyngeal aspirate specimens were collected from those patients and common respiratory virus antigens including influenza virus A and B, respiratory syncytial virus, adenovirus, parainfluenza virus types I, II, and III were tested by indirect immunofluorescent assay. The patients with atypical pneumonia diagnosed by X-ray and evidences of common respiratory virus infection were admitted to the regular ward for children with respiratory diseases. Children with pneumonia demonstrated by X-ray and negative for common respiratory viruses were admitted to the isolated ward for suspected SARS patients for the first step and further viral etiological studies were requested. RT-PCR was performed for those patients to detect gene fragments of human metapneumovirus (HMPV), rhinovirus (RhV) and enterovirus (EV) in their specimens. Nested RT-PCR was also developed to detect SARS coronavirus gene fragment from the specimens. Primer sequences for SARS virus detection with the PCR were selected according to the primer sequences published online by WHO on April 18, 2003. All the primers derived from the sequence at the 1b frame of coronavirus replicase gene and products with a size of 368 or 348 bp were expected with 2 different primer pairs.

RESULTS: Amplicons with the sizes of 368 bp and 348 bp were obtained from a throat swab specimen collected from a 17 years old girl, who was admitted to the isolated ward because of high fever (39.5 degrees C) for 7 days, cough for 2 days, low WBC count, and pneumonia shown by X-ray when she visited the "Fever and Cough" Clinic, and without known history of contact with probable SARS patient. Antigens for the common respiratory viruses were all negative, RT-PCR for HMPV, RhV and EV were also negative while RT-PCR with different primer pairs for SARS virus were all positive which indicated that SARS coronavirus gene fragments were amplified from the specimen from this girl. The amplified fragment with a size of 368 bp was sequenced and the sequence was compared with those in the GenBank. The sequence shared 100% homology with the sequences from 1b frame of replicase genes from all 17 of SARS coronaviruses published in the GenBank so far, and shared very low homology with 2 reference strains of human coronavirus as well as other animal coronaviruses. The serum collected before her discharge from the hospital (19 days after the onset of the disease) showed SARS specific IgM and IgG antibodies.

CONCLUSION: These data indicate that the patient was a confirmed case of SARS. It is of great importance to differentiate SARS patients from those infected with common respiratory viruses during SARS epidemic, especially for pediatric patients, because most of the patients visiting the outpatient department present with the symptoms of fever, cough and normal WBC count. The data mentioned above indicate that antigen and gene detections for those common respiratory viruses are useful methods for the differentiation to avoid the spread of SARS.

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