Table 1.

Considered retrospective studies that found evidence for an early SARS-CoV-2 circulation

Reference	Methods	Samples	Study period and location	Findings	Caveats (#) and confirmations (§)
25	Nested-PCR, Sanger sequencing	39 oropharyngeal swabs collected within the measles/rubella surveillance system	Sep 2019–Feb 2020 Lombardy (Italy)	Viral RNA detected in Dec 2019	# The use of nested-PCR increases the chances of false positives # Single methodological approach § The positive sample was confirmed by sequencing § Samples were analysed in a follow-up study24
24	Nested-PCR, Sanger sequencing, commercial ELISA, neutralisation assay	Oropharyngeal swabs, urine and sera (N=435) collected within the measles/rubella surveillance system	Aug 2018–Apr 2021 Lombardy (Italy)	Viral RNA detected since 12 Sep 2019. The first positive patient was also IgG and IgM positive	# The use of nested-PCR increases the chances of false positives § Positive samples were confirmed by sequencing, and different variants were found § Samples from 100 patients collected before the first case were RNA negative § Follow-up studies with an external laboratory are currently ongoing
26	Immunohistochemistry, in-situ hybridisation	One skin biopsy from a patient with dermatosis	Nov 2019 Milan (Lombardy, Italy)	Presence of SARS-CoV-2 in paraffin samples	# Viral RNA undetected with RT-PCR technology # Results not confirmed by another laboratory § Positivity was confirmed by two different techniques on paraffin sections
21	Nested-PCR, RT-PCR	40 composite wastewater samples collected within the framework of wastewater-based environmental monitoring	Oct 2019–Feb 2020 Milan (Lombardy), Turin (Piedmont), Bologna (Emilia Romagna) (Italy)	15 samples were confirmed positive by both methods. The earliest detections were on 18 Dec 2019 (Milan and Turin) and 29 Jan 2020 (Bologna)	# The use of nested-PCR increases the chances of false positives # Results not confirmed by another laboratory § Positivity confirmed by RT-PCR and sequencing § The plants where the positive samples were collected serve a population of approximately 4 million inhabitants
20 49	Contact tracing from Directorate General for Health, Lombardy Region	Official records	Jan 2020–Feb 2020 Lombardy (Italy)	Epidemiological investigations suggested a sustained transmission across all Lombardy provinces from 1 Jan 2020	# Uncertainties due to difficulties in identifying the correct epidemiological links between cases # No data were available to distinguish imported versus local infections
22	In-house ELISA, virus neutralisation assay	959 blood samples from asymptomatic individuals enrolled in a prospective lung cancer screening trial	Sep 2019–Mar 2020 Italy	RBD-specific Ig in 11.6% samples since Sept 2019 (14%); cluster of positive cases (>30%) in the 2nd week of Feb 2020. Highest positive rate (53.2%) in Lombardy.	# No samples before September 2019 were analysed # Ig detection is less specific, and a proportion of cases could be attributed to false positivity § Six samples also showed neutralising activity § Results for a subsample confirmed in a follow-up study23
23	ELISA, microneutralisation assay	29 plasma samples	Jul 2019–Feb 2020 Italy	Presence of IgM and IgG antibodies in the pre-pandemic period	# Ig detection is less specific, and a proportion of cases could be attributed to false positivity § Results partially confirmed by an external laboratory
13	Commercial ELISA, in-house virus microneutralisation assay	Serum samples collected from 9144 adults from a French general population-based cohort	Nov 2019–Mar 2020 France	3.9% of samples were positive to anti-SARS-CoV-2 IgG test and 13 had neutralising activities	# No samples before November 2019 were analysed # Ig detection is less specific, and a proportion of cases could be attributed to false positivity # Results not confirmed by another laboratory § Thirteen samples showed neutralising activity
14	RT-PCR	14 respiratory samples collected from patients hospitalised in the intensive care unit	Dec 2019–Jan 2020 France	Confirmed diagnosis of SARS-CoV-2 infection in one patient with haemoptysis in Dec 2019	# Positivity was not confirmed by sequencing # Single methodological approach # Results not confirmed by another laboratory § The result was confirmed with a second RT-PCR protocol and by different laboratory operators
12	Flow cytometry-based method	101 blood samples from uninfected individuals	May 2019 UK	Presence of pre-existing antibodies recognising SARS-CoV-2 in uninfected individuals	# Ig detection is less specific, and a proportion of cases could be attributed to false positivity # Results not confirmed by another laboratory § Two donors presented simultaneously antibodies of the three classes
11	RT-PCR, SISPA, metagenomics	Six human sewage water samples	Oct 2019–Mar 2020 Santa Catarina (Brazil)	Viral RNA detected since 27 Nov 2019	# No clinical records of local COVID-19 cases in 2019 # The sewer where the samples were collected serves a population of approximately 5000 inhabitants § Positive samples were confirmed by sequencing § Virus detection was confirmed by an independent laboratory
10	Pan-Ig ELISA, microneutralisation, ortho total Ig S1 ELISA, RBD/ACE2 blocking activity assays	7389 serum specimens from blood donations	Dec 2019–Jan 2020 California, Connecticut, Iowa, Massachusetts, Michigan, Oregon, Rhode Island, Washington, Wisconsin (USA)	1.4% of samples were reactive by pan-Ig, 84 of 90 had neutralising activity. Presence of anti-SARS-CoV-2 reactive antibodies since 13–16 Dec 2019.	# No samples before Dec 2019 were analysed # Ig detection is less specific, and a proportion of cases could be attributed to false positivity # Results not confirmed by another laboratory § The majority of Ig-positive samples showed neutralising activity

Ig, immunoglobulin; RBD, receptor-binding domain of SARS-CoV-2; RT-PCR, real-time PCR; S1, subunit 1 of the spike protein of SARS-CoV-2; SISPA, sequence-independent single-primer amplification.