Detection of SARS-CoV-2 virus using acute respiratory infections sentinel surveillance system (PIDIRAC) in Catalonia (Spain)

In the context of the Covid-19 pandemic in Catalonia (Spain), the rst SARS-CoV-2 case conrmed was notied to the Catalonia Epidemiological Surveillance Network (RVEC) on 25 February 2020. The present study describes and analyses the respiratory samples obtained in primary care using PIDIRAC epidemiological sentinel surveillance system to complement the pandemic surveillance system activated, and describe whether SARS ‐ CoV-2 was circulating before the rst case detected in Catalonia, between October 2019 and April 2020. During this period, 878 respiratory samples from patients with acute respiratory infection or inuenza syndrome obtained by PIDIRAC epidemiological sentinel surveillance system were analysed. Of the total sample, 51.9% tested positive to inuenza virus and 48.1% to other respiratory viruses, with SARS-CoV-2 being present in 6 samples. The rst SARS ‐ CoV ‐ 2 positive case showed the rst symptoms on 2 March 2020. These were 3 men and 3 women aged between 25 and 50 years old (mean age 44.5 years old). Fever, general discomfort, coughing, chills, and arthromyalgia were the most frequent symptoms in the SARS-CoV-2 cases. Likewise, 44 tested during They were typed as none of them being The acute respiratory infections sentinel surveillance system (PIDIRAC) reinforces the global epidemiological surveillance, allows to corroborate whether there is virus circulation or not, and helps conrm that generalised community transmission in Catalonia took place in mid-March.


Introduction
On 31 December 2019, the Wuhan Municipal Health Commission in the Province of Hubei, China, announced a cluster of 27 cases of pneumonia of unknown aetiology, including 7 severe cases, with common exposure taking place in a wholesale market of seafood, sh and live animals in the city of Wuhan, without identifying the outbreak source ( 1 ). The market was shut down on 1 January 2020. On 7 January 2020, Chinese authorities identi ed a novel type of virus from the Coronaviridae family as the causative agent of the outbreak, which was called coronavirus SARS-CoV-2 ( 2 ). The Chinese authorities shared its genetic sequence on 12 January 2020. On 30 January 2020, the World Health Organization declared the outbreak of coronavirus SARS-CoV-2 a public health emergency of international concern in China ( 3 ). Afterwards, the outbreak spread beyond China, affecting other countries, many of them in Europe. In February, the number of cases started to fall in China as it increased in other countries, especially in Europe ( 4 ).
Reported cases increased dramatically and spread exponentially around the world. On 11 March 2020, WHO declared the infection of SARS-CoV-2 a pandemic ( 5 ).The outbreak in Italy affected a large number of cases, and from there, the cases spread to Spain. In Catalonia as to 27 May 2020, the number of con rmed cases was 58,480 people ( 6 ).
The rst con rmed case of SARS-CoV-2 in Catalonia was noti ed on 25 February 2020 to the Catalonia Epidemiological Surveillance Network (RVEC). It was a 36-year old Barcelona female resident who had travelled from 12 to 22 February to the cities of Bergamo and Milano. Symptoms appeared on 20 February; she required hospitalization and evolved positively.
Cases increased slowly in Catalonia during the containment stage -rst stage of the pandemic-as RVEC strictly applied the action protocol for cases of infection with the new coronavirus SARS-CoV-2 drawn up by the Sub-directorate General for Surveillance and Public Health Emergency Response ( 7 ) -agreed upon as part of the State Epidemiological Surveillance Network (RENAVE)-and by the Coordination Centre for Sanitary Alerts and Emergencies (CCAES) ( 8 ). A screening system was speci cally established by the RVEC and the Catalonia Epidemiological Surveillance Emergency Service (SUVEC) with the information provided by health care centres and the laboratory network from both the public and private health care sector. This is an active surveillance of cases and contacts to establish the necessary tests and implement isolation and quarantine required, which implied the restriction of movement of the cases' close contacts. This was a pioneering measure as Catalonia kept a limited transmission of the located chains for some time before reaching community transmission and helped limit transmission until the mitigation stage with generalised community transmission on 14 March 2020.
In addition to the active surveillance system established as a response to the health alert, Catalonia has implemented a surveillance system for in uenza and other acute respiratory infections (PIDIRAC) since the 1999-2000 season. It is based on a surveillance network with 56 primary care physicians, family physicians and paediatricians who work in 44 primary care health care centres homogenously distributed, covering around 1% of Catalonia population. The PIDIRAC system is part of the national in uenza surveillance system. For many years ( 9 , 10 ), it has been supported by several systems and information sources from different territories around the country, being also part of the European In uenza Surveillance Network (EISN) which is coordinated by the European Centre for Disease Control (ECDC) since 2008, contributing in this way to the overall objectives in terms of in uenza international surveillance. The EISN also encompases several laboratories that are part of the European Reference Laboratory Network for Human In uenza (ERLI-Net).
The objective of this study is to describe and analyse the respiratory samples obtained in primary care as part of the epidemiological sentinel surveillance system PIDIRAC, to complement the active surveillance system implemented in the pandemic and establish whether SARS-CoV-2 was circulating before the rst case was detected in Catalonia, between October 2019 and April 2020.

Surveillance system
In January 2020, Catalonia deployed a reinforced surveillance system in the light of the public health emergency of international concern, in collaboration with RENAVE and CCAES. During the containment stage, and following WHO guidelines, the clinical and epidemiological criteria of the Action protocol for cases of infection of the new coronavirus SARS-CoV-2 (7) was implemented. Clinical and epidemiological criteria changed with the evolution of the infection and the pandemic stage. Once the rst local cases emerged in Italy on 21 February 2020 ( 11 ), a case under investigation was considered to be any individual with a clinical picture compatible with acute respiratory infection of sudden onset and some of the following symptoms: coughing, fever, dyspnoea regardless of its severity and who, in the 14 days prior to symptoms onset, met any of the following epidemiological criteria: travel history to areas with evidence of community transmission such as China (all provinces, including Hong Kong and Macao), South Korea, Iran, Japan, Singapore and Italy (regions such as Lombardy, Veneto, Emilia-Romagna, Piedmont). Cases under investigation were RT-PCR tested for SARS-CoV-2 using nasopharyngeal swabs.
Additionally, other cases outside these areas, exposure to events with a cluster of cases or with transmission chains of signi cant size as well as with a history of close contact with a possible or con rmed case were also researched.
Also, PIDIRAC continued its activity with professionals who collected pharyngeal and nasal samples during October 2019-March 2020 surveillance season. Besides in uenza A, B, and C virus, other virus under study include respiratory syncytial virus, parain uenza virus, adenovirus, coronavirus, enterovirus, rhinovirus, metapneumovirus and bocavirus.
Sentinel physicians notify the cases or cluster of cases that are highly likely to have respiratory infection and collect respiratory samples that are sent for diagnosis to Hospital Clínic de Barcelona laboratory, a reference laboratory for respiratory virus in Catalonia.

Microbiology
All the samples collected as part of PIDIRAC sentinel surveillance were part of a prospective study on in uenza A, B and C virus, adenovirus, respiratory syncytial virus, bocavirus, metapneumovirus, parain uenza virus, rhinovirus, enterovirus and coronavirus (CoV) using a multiple Reverse Transcriptase PCR (RT-PCR) in real time.
The respiratory virus were analysed with a semiautomated platform (Flow system, Roche Diagnostics GmbH, Mannheim, Germany) that includes nucleic acids extraction with MagnaPure and ampli cation and detection of the virus using thermocycling LightCycler 480. Speci c primers and probes were used for the above mentioned virus (LightMix® Modular, TIB MOLBIOL, GmbH, Berlin, Germany). All positive samples for coronavirus were subsequently typed for HKU1, OC43, NL63, 229E. Every respiratory sample was frozen at -80ºC and retrospectively studied for the presence of SARS-CoV-2 with the cobas SARS-CoV-2 assay on the 6800 platform (Roche Diagnostics). The cobas assay amplify and detect two viral targets: ORF1 a/b, a non-structural region that is unique to SARS-CoV-2 and a conserved region in the Egene, which is a structural protein envelope for pan-Sarbecovirus detection.

Statistical analysis
For each case, the following variables were analysed: age, sex, municipality, date of case diagnosis and noti cation, clinical picture, risk factors, hospitalization, ICU admission, travel history, close contact of COVID19 and whether it was a health care professional. A descriptive study has been done and the presence of signi cant differences in these variables between SARS-CoV-2 cases and the rest of coronaviruses has been analysed in a bivariate study with Chi2 test, with statistical signi cance at 95%.

Ethical considerations
All data used in the analysis were collected during routine public health surveillance activities, as part of the legislated mandate of the Health Department of Catalonia, the competent authority for the surveillance of communicable diseases, which is o cially authorized to receive, treat and temporarily store personal data on cases of infectious disease. Therefore, all study activities formed part of public surveillance and were thus exempt from institutional board review and did not require informed consent. All data were fully anonymized. March 2020. In 4 cases (66.7%), patients with SARS-CoV-2 showed a sudden onset of symptoms while 12 patients (41.4%) affected by other coronavirus had a sudden debut. Fever and dyspnoea were more frequent in the SARS-CoV-2 cases (100% and 33.3%, respectively) compared to other CoV (72.4% and 6.9%, respectively), although without signi cant differences between the two groups. Symptoms shown are included in Table 2. None of the SARS-CoV-2 cases had been in contact with a possible case of in uenza or SARS-CoV-2 nor did they have travel history to areas with community transmission at that moment. One of the SARS-CoV-2 cases was a health care professional.

SARS-CoV
All patients evolved favourably. None of the cases affected with non-SARS CoV required hospitalization. One of the cases with SARS-CoV-2, with previous respiratory pathology, required hospitalization and evolved favourably.

Discussion
In the case of a pandemic like SARS-CoV-2, considering different epidemiological surveillance systems is essential to reinforce monitoring and early detection of circulating cases as much as possible in order to implement different public health actions depending on the required stage.
In Catalonia, the rst Covid-19 case was detected on 25 February. From that moment until mid-March, transmission chains of the virus were perfectly located during the containment stage and cases were immediately studied and followed-up to contain the disease (7). The PIDIRAC system, functioning since October 2019, corroborated that although the regular seasonal coronavirus was already circulating, SARS-CoV-2 community presence started at the beginning of March, which indicated that transmission increased from that moment onwards. Likewise, this study allows to conclude that the PIDIRAC system did not detect any case prior to the rst RVEC noti cation.
The rst six SARS-CoV-2 cases reported through the PIDIRAC sentinel network were mild cases detected during the containment stage, after the rst SARS-CoV-2 case was detected by RVEC.
Despite the absence of signi cant differences between SARS-Cov2 and the other CoV, SARS-Cov-2 mainly affects adults with risk factors while CoV affected almost all age groups and, to a lesser degree, patients with risk factors. None of the SARS-Cov-2 cases presented coinfection with other respiratory virus under study unlike CoV cases which presented coinfection with other different respiratory virus (almost one third of the cases). Similarly, Gaunt ER et al in UK describe coinfection between CoV and RSV, in uenza and adenovirus between 11 and 41% ( 12 ).
Following WHO recommendations, both surveillance systems -sentinel and pandemic containment systems-coexisted in Catalonia. Likewise, European countries progressively implemented a strong surveillance of COVID-19 cases to draw a global strategy to contain COVID-19, which was progressively complemented with different epidemiological surveillance systems (3,13 ).The sentinel surveillance systems are recommended by the European surveillance network, especially in European regions or states where mild cases were not tested at the beginning, as it is our case ( 14 ).
France was the European country where the rst three cases were diagnosed on 24 January. These were people with travel history to Wuhan ( 15 ). Likewise, their acute respiratory infections sentinel surveillance system showed that several nasopharyngeal swabs tested positive to SARS-CoV-2. Excess cases due to acute respiratory infection was quanti ed in France at the beginning of March compared to the number of visits expected from the seasonal in uenza virus epidemic ( 16 ).
Regarding the study limitations, it is worth noting that the respiratory clinical portrait of the in uenza in the context of the PIDIRAC Program could not allow for identifying a COVID-19 possible case, either because symptoms were very mild and the person did not visit the physician or because it was an asymptomatic COVID-19 case. The presence of pathogens others than SARS-CoV-2 does not guarantee that a patient was not SARS-CoV-2 positive ( 17 ). However, our study did not show any coinfection with other virus. Fabiona Gámbaro et al demonstrates that the virus had been previously circulating in France, with the additional challenge of asymptomatic cases ( 18 ). Similarly, as the sentinel surveillance system is focused on community, it would have not detected severe in uenza syndrome cases that would have been hospitalised prior to the announcement of the international public health emergency at the end of January. However, we corroborated that the other regular epidemiological surveillance systems, i.e. the compulsory declaration diseases system (MDO) and Catalonia microbiological noti cations system (SNMC), did not detect any case before 25 February.
The acute respiratory infections sentinel surveillance system, PIDIRAC, reinforces the global epidemiological surveillance, allows for con rming whether the virus is circulating or not, and helps verify that generalised community transmission in Catalonia took place in mid-March. Public Health departments should rely on sentinel systems to determine SARS-CoV-2 community transmission levels and thus prioritise community mitigation measures ( 19 ). Marissa L et al gathers that COVID-19 case identi cation through a sentinel surveillance system helps con rm SARS-CoV-2 community transmission and that the implementation of early community measures is key for much effective mitigation of SARS-CoV-2 transmission ( 20 , 21 ). As we approached generalised SARS-CoV-2 circulation, surveillance based on clinical criteria and sample collection by sentinel physicians was essential to assess the situation ( 22 ). PIDIRAC sentinel system is useful for monitoring SARS-CoV-2 community transmission and geographic expansion as well as being alert and prepared in the case of a potential emergency that may arise from a virus onset with pandemic potential, as that of in uenza, while the COVID-19 pandemic remains active

Author contributions
All the authors have been directly involved in the article untitled: Detection of SARS-CoV-2 virus using acute respiratory infections sentinel surveillance system (PIDIRAC) in Catalonia (Spain). In addition, all of them have substantially contributed in all the necessary information described in the manuscript, from the conception and design, the acquisition and data processing, the analysis and its interpretation. All authors have contributed in a critical revision of the contents.

Con icts of interest
The author declares that neither they nor any of the co-authors have a con ict of interest regarding the publication of this manuscript.

Ethical approval:
This article does not contain any studies with human participants or animals performed by any of the authors.

Ethical considerations
All data used in the analysis were collected as part of routine public healthsurveillance activities and were therefore exempt from institutional review board review.  Respiratory virus in the population during winter season. Catalonia, October 2019-April 2020