Serological investigation of SARS-CoV-2 infection in patients with suspect measles, 2017–2022

image

Several studies investigated the pre-pandemic circulation of SARS-CoV-2 and concluded that the virus was likely already circulating outside of China before the first outbreak observed in Wuhan [3]. While PCR-based tests allow to directly detect the virus, false positive results can be obtained with serological assays as cross-reacting antibodies can be present in sera of uninfected patients. Therefore, large-scale investigations analyzing samples collected for some time before COVID-19 emergence would be required to observe a rise in positivity rates consistent with initial viral circulation. Unfortunately, such investigations are still lacking.

We tested for anti-SARS-CoV-2 IgG all sera received for measles/rubella surveillance collected from measles and rubella virus-negative patients. Indeed, patients with COVID-19 can develop morbilliform rash [6, 10, 11] and our previous investigations demonstrated that both SARS-CoV-2 RNA and antibodies against this virus can be detected in these patients [5]. IgG were chosen because the available detection assay was characterized by a high specificity (99.6%). Additionally, not all patients produce IgM, and their level decreases a few days after symptom onset. Although there is individual variation, IgG against SARS-CoV-2 develop ~ 2 weeks after infection, when the virus may no longer be detectable, and remain measurable for a long time [12]. Similarly, cutaneous manifestations appear later during SARS-CoV-2 infection, up to 4 weeks after the onset of respiratory symptoms [10, 11], with a timeline more consistent with IgG levels.

Thirteen patients were positive for anti-SARS-CoV-2 IgG and seven of them were symptomatic during the pre-pandemic period. Given the low number of investigated samples and identified positives, we compared the percentage of detected positive samples to the expected proportion of false positive results (0.04%) and observed that the difference between these two proportions was statistically significant for the year 2019, including when considering the first and second half separately, but not for the years 2017 and 2018. Additionally, in 2019, the percentage of IgG-positive patients was significantly lower among SARS-CoV-2 RNA-negative patients than among SARS-CoV-2 RNA-positive patients.

The semi-quantitative method we used showed that the strength of the signal increased in 2021–2022. Although weakly reacting antibodies could indicate a false positive signal, highly reacting antibodies identified in a few patients symptomatic in later phases of the pandemic (2021–2022) could have been determined by re-exposures or vaccination. Indeed, three of the five strongly positive samples were collected after COVID-19 mass vaccination campaigns had started and all five were sampled after at least one year of virus circulation.

The highest percentage of IgG positivity during the pre-pandemic period was during the second half of 2019, with three positive samples in September and October. This coincided with an increase in negativity for measles and a widening of the peak of the number of discarded cases per 100,000 inhabitants, which followed a measles epidemic that ended in July. In other words, while measles virus stopped circulating, there was still a higher-than-normal number of patients experiencing fever and rash. This is consistent with the observed increase in measles discarded rate for the year 2019 [5]. Noticeably, this is also the time when we detected the first SARS-CoV-2 RNA-positive sample (September 12th, 2019, [5]), and it may be that this is the moment when SARS-CoV-2 started circulating in Lombardy. These results are in agreement with other studies identifying IgG and neutralizing antibodies against SARS-CoV-2 in the same time period in Europe [3, 4, 9].

This study has some limitations. Firstly, the low number of available samples limits our findings and the power of our statistical analyses. Secondly, the investigated period is limited to 3 pre-pandemic years and 3 pandemic years with a variable number of samples in each period. However, the population type was consistent across the years of the study. Additionally, confirmation assays, such as neutralization, was not performed and, while the specificity of the used assay is high, the kit is characterized by a low sensitivity (47.3% and 94.4% before and after day 10 after symptom onset, respectively) and some cases may have gone undetected. Nonetheless, although this study cannot conclusively establish when the virus started circulating in Lombardy, it should serve the purpose of stimulating further research as evidence for undetected SARS-CoV-2 pre-pandemic circulation continues to accumulate.

Leave a Reply