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.