Detection of 2019 novel coronavirus (2019-nCoV) in patients with inuenza-like illness (ILI)

2019 novel coronavirus (2019-nCoV) is �rstly found in Wuhan and has caused over 14,000 infections all over the world. We retrospectively investigated the presence of 2019-nCoV among inuenza-like illness patients in Wuhan. Nine cases in January 2020 were 2019-nCoV positive, suggesting the virus has established community transmission in Wuhan, the origin and center of this epidemic. Authors Wen-Hua Kong, Yao Li, and Ming-Wei Peng contributed equally to this work.


Introduction
In late December 2019, clusters of patients with pneumonia of unknown etiology that were epidemiologically linked to a seafood and wet animal wholesale market were reported in Wuhan, Hubei Province, China 1 .The causative pathogen of the pneumonia clusters was soon identi ed as a novel coronavirus which shared 79.5% sequence identity to the severe acute respiratory syndrome coronavirus (SARS-CoV) 1,2 and was named 2019 novel coronavirus (2019-nCoV).Since then, the virus has spread into all the provinces of China and 23 other countries 3 .As of February 1, 2020, more than 14,000 cases of 2019-nCoV infection have been con rmed in China and 4109 cases of them were reported in Wuhan.
As the China CDC declared, the community transmission of 2019-nCoV has established in the city of Wuhan and nearby regions 4 .However, the clinical outcome of 2019-nCoV not only includes viral pneumonia like SARS-CoV, but also covers mild illness and even asymptomatic infection 5,6 .The existing medical resources cannot support the nucleic acid test of a large number of mild cases 4 and reliable serological assay is still not commercially available.The whole picture of 2019-nCoV epidemic in Wuhan remains unclear.

The Study
In order to better understand the current epidemic in Wuhan, particularly the status of mild illness cases, we retrospectively investigated the presence of 2019-nCoV among local in uenza-like illness (ILI) patients who visited hospital between October 6, 2019 and January 21, 2020.Sustained surveillance for ILI cases and their etiology has been implemented in Wuhan since 2005, based on the National In uenza Surveillance Network of China 7 .According to the latest National In uenza Surveillance Plan, a patient is identi ed as ILI-case if s/he has the sudden onset of a fever >38℃ and cough or sore throat 8 .In the two national in uenza sentinel hospitals, the Children's Hospital of Wuhan and Wuhan No. 1 Hospital, the number of ILI cases and total outpatient number are collected weekly.Clinical samples of ILI patients are also collected, if the patient had a fever for less than 3 days and has not been treated with antiviral drugs.
After verbal informed consent was obtained from parents or caretakers, throat swabs were collected from ILI patients in 3.5 ml viral transport medium and were delivered to Wuhan Center for Disease Prevention and Control for laboratory diagnosis of in uenza virus.Each sentinel hospital is required to collect 20 ILI samples every calendar week.
A total of 640 throat swabs were collected from ILI patients in the 16-week period between October 6, 2019 and January 21, 2020 (2019W40 to 2020W03).We performed 2019-nCoV ORF1ab/N qPCR assay (BioGerm, Shanghai, China) on the nucleic acids extracted from these specimens.The nucleic acids have been used in the detection and subtyping of in uenza virus 8 .They were initially extracted from 200μl of throat swabs using a PANA9600E automated nucleic acid extraction system (Tianlong, Xi'an, China) and were stored at -70℃.All assays have been established in our laboratory and procedures were performed following the manufacturers' instructions.
The time period in concern coincided with the winter peak of in uenza and other respiratory illnesses.The ILI case numbers in all age groups have increased dramatically since early December and reached the peak by the New Year (Fig. 1).Particularly, the 5-14 years group presented an increment over 24-folds (2019W40-W47: 75 cases/week; 2019W52: 1916 cases).The percentage of ILI patients in all outpatients experienced a similar rise: the average percentage was 1.07% during 2019W40 to W47, and soared up to 9.44% in 2020W01.
The ILI patients involved in this study comprised 315 males and 325 females, ranging from 9 months to 87 years (median age 8 years, mean age 22.7 years).The 2019-nCoV RNA was detected in 9 patient specimens (Table1, Supplementary Table 1).All of them were collected in January 2020 (2020W1-2020W3), when the seasonal in uenza (mainly A/H3 and B/Victoria) remained to be active, yet no coinfection has been detected.
The basic demographic information and illness timeline of nine 2019-nCoV infected patients is listed in Table 2. Their gender ratio was 1.25 (5 males vs. 4 females) and all of them were adults (age range: 35-71 years).These demographic features are consistent with other available reports about 2019-nCoV patients 6,9 .The onset date of the earliest case was January 4, 2020, one week after the outbreak was rst reported by hospital 9 .Although the weekly sample size was rather small, it seems that 2019-nCoV infection was gradually expanding among ILI cases during January.In the last week of observation, the positive rate of 2019-nCoV has exceeded that of in uenza virus among the group over 25 years (Table 1).
Interestingly, nine 2019-nCoV patients came from 6 different districts of Wuhan metropolitan and surrounding areas, which provided additional evidence for the community transmission in Wuhan.

Conclusions
Our work provides context for understanding the early stage of the novel coronavirus epidemic.Recent epidemiological studies estimated that the basic reproductive number of 2019-nCov was 2.2 to 2.68, and the epidemic doubling time was 6.4 to 7.4 days 9,10 .The occurrence and expansion of 2019-nCoV among ILI patients in Wuhan are consistent with those assessments.Given the fact that community-acquired 2019-nCoV infection cases have been reported outside Hubei 11 , we suggest strengthening pathogen surveillance of ILI cases in major Chinese cities and other regions in the world.11Pei, P. Shenzhen con rms community transmission coronavirus cases, <http://www.chinadaily.com.cn/a/202002/02/WS5e36c08ba310128217274349.html>(2020).Figures

Table 2 .
The demographic information and illness timeline of nine 2019-nCoV infected ILI patients in Wuhan.