Italian study finds SARS-CoV-2 in clinical samples collected before December 2019

By Suchandrima BhowmikAug 30 2022Reviewed by Benedette Cuffari, M.Sc.

A new Environmental Research journal research paper discusses the association of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with morbilliform skin rash. These findings also provided evidence of the early circulation of SARS-CoV-2 in Northern Italy prior to the start of the coronavirus disease 2019 (COVID-19) pandemic.

Study: Molecular evidence for SARS-CoV-2 in samples collected from patients with morbilliform eruptions since late 2019 in Lombardy, northern Italy. Image Credit: OneSideProFoto / Shutterstock.com

Background

In December 2019, SARS-CoV-2 was first isolated in China following reports of hospitalized patients experiencing atypical pneumonia. Soon after, molecular evolutionary experiments revealed that SARS-CoV-2 likely spilled over to humans from a bat reservoir; however, its original spillover mechanism remains debatable.

Despite the lack of a definitive timeline on when SARS-CoV-2 initially emerged, previous evolutionary studies indicate that the virus likely circulated in China for several months before the first outbreak was recorded in Wuhan, China. Soon after, an increasing number of cases were reported in several European and North American countries by mid-January 2020.

Italy was the first European country to report sustained SARS-CoV-2 community transmission. This country subsequently became the epicenter of the epidemic in Europe, with Lombardy being the most affected.

The SARS-CoV-2 strain that circulated in Lombardy, as well as much of Europe soon after its initial detection in Italy, differed from the Wuhan-Hu-1 strain, which was the reference genome originally identified in China. In fact, some of the different mutations present within this strain included A23403G (Spike D614G), C14408T (RdRp P323L), and C3037T (synonymous). This strain, which has since been named B.1 in Pangolin and 20A in NextStrain, is often referred to as the DG1111 haplotype and comprises an αβ mutational signature.

Several studies suggest that SARS-CoV-2 was circulating in many nations prior to its official detection. In fact, SARS-CoV-2 ribonucleic acid (RNA) has been detected in wastewater samples obtained in Brazil and Northern Italy in 2019.

SARS-CoV-2 was also detected in the respiratory samples of a French patient with hemoptysis, the lungs and blood of a patient in Milan who died of acute circulatory insufficiency, and oropharyngeal swabs of a Lombardy child with suspected measles in December 2019. Viral RNA and antigens have also been detected in paraffin-embedded skin biopsies of women in Milan with dermatosis in November 2019.

Several skin manifestations have been reported in patients diagnosed with COVID-19. These manifestations can occur at any stage of the disease.

In addition to the variable duration, prognosis, and severity of these dermatological effects, these symptoms can even occur in the absence of the common respiratory symptoms of COVID-19, thus causing diagnosis to be even more challenging.

In 2019, suspected measles and rubella cases were two times higher as compared to the average of the previous two years. Moreover, the percentage of suspected cases that tested negative increased from 30% to 70% during the first wave of the pandemic and 100% by 2021.

The increased incidence of both measles and rubella, combined with the skin manifestations associated with COVID-19, led the researchers of the current study to investigate whether COVID-19 could cause morbilliform skin eruptions.

About the study

The current study was conducted by the Network of Italian Reference Laboratories for Measles and Rubella (MoRoNet)- and World Health Organization (WHO)-accredited Subnational Reference Laboratory for measles and rubella surveillance.

A total of 435 urine, serum, and oropharyngeal samples were collected from 156 patients tested for SARS-CoV-2. All samples included in the current analysis were negative for both measles and rubella.

Samples from 44 cases were collected between August 2019, which coincided with the unexpected increase in suspected measles/rubella cases, as well as late February 2020, which were considered pre-pandemic cases.

Twelve samples were also collected between March 2020 and March 2021 and were referred to as pandemic cases. Additionally, samples collected from 100 cases between August 2018 and July 2019 were used as controls.

A total of 289 urine and oropharyngeal samples were analyzed for the presence of SARS-CoV-2 RNA followed by sequence analysis. Sera from 38 pre-pandemic, 98 control, and 10 pandemic cases were analyzed for anti-SARS-CoV-2 immunoglobulin A (IgA), IgM, and IgG.

Plaque reduction neutralization assays were used to determine neutralizing antibody titers.

Study findings

Positive SARS-CoV-2 infection was identified in 11 pre-pandemic and two pandemic cases. Of these 13 subjects, viral RNA was detected in urine and respiratory samples for six and seven patients, respectively. Notably, viral RNA was detected in the urine of only pandemic cases.

Of the 11 pre-pandemic samples that tested positive for SARS-CoV-2, nine were collected in 2019. The earliest SARS-CoV-2-positive urine sample was collected on September 12, 2019, from an eight-month-old child who also had detectable serum levels of SARS-CoV-2 IgG and IgM.

None of the SARS-CoV-2-positive samples were positive when the Real-Time polymerase chain reaction (PCR) diagnostic protocol was used. This indicates that all samples exhibited low viral loads that were less than the detection threshold level.

Four pre-pandemic samples were positive for anti-SARS-CoV-2 antibodies, with IgM most frequently detected. Only one of these serum samples contained partially neutralizing antibodies.

Conversely, neutralizing antibodies were detected in two pandemic samples. However, both of these samples were SARS-CoV-2 RNA-negative.

Twelve samples collected before the first aforementioned SARS-CoV-2-positive sample was collected on September 12, 2019, were positive for IgA, IgM, and IgG. Only four of these samples exhibited partial neutralization.

Early pre-pandemic samples that tested positive for SARS-CoV-2 were mostly localized Brescia and Milan, with most Milanese cases localized in north-western Milan. All pandemic samples were also localized in the province of Milan.

Out of the 11 pre-pandemic cases, five were collected between October 12, 2019, and October 23, 2019, whereas all pandemic samples were collected during early 2021.

Most of the viral sequences were obtained from pre-pandemic samples and exhibited several major mutations including C3037T, A23403G, and C14408T that belonged to the β group of mutations. Pre-pandemic SARS-CoV-2 strains are believed to be approximately six mutations away from the SARS-CoV-2 progenitor strain and belong to the αβ lineage, which has produced all major circulating strains to date.

Conclusions

The current study confirms that SARS-CoV-2 was circulating in Northern Italy by late 2019, as confirmed by the presence of SARS-CoV-2 genetic material in several urine and oropharyngeal swab samples. Importantly, none of the samples that tested positive for SARS-CoV-2 were obtained from patients with a history of international travel.

The study findings also suggest an association between morbilliform eruptions and SARS-CoV-2 infection. However, future studies are needed to better understand the relationship between this type of skin rash and COVID-19.

Future retrospective studies in areas with early SARS-CoV-2 circulation that utilize metagenomic methods are needed to accurately determine the evolutionary history and time of SARS-CoV-2 emergence.