Spread of multiple SARS-CoV-2 lineages April-August 2020 anticipated the second pandemic wave in Lombardy (Italy).

During the early phase of the pandemic (20 February-4 April 2020), we have investigated the temporal and geographical evolution of the virus in Lombardy showing the circulation of at least seven lineages distributed differently in the Region. In the present study, the molecular epidemiology of SARS-CoV-2 was monitored in a period between two pandemic waves in order to track the circulation of new variants (April-August 2020). A great majority of SARS-CoV-2 strains (70.8%) belonged to lineages B, B.1, B.1.1 and B.1.1.1, and five strains belonging to four lineages were already reported in Italy (B.1.1.148, B.1.1.162, B.1.1.71, and B.1.425). In addition, 21 SARS-CoV-2 strains belonged to six lineages not previously observed in Italy were detected. No variants of concern were observed. A total of 152/1274 (11.3%) amino acid changes were observed among spike gene sequences and only 26/152 (17.1%) occurred in the receptor-binding domain region of the spike protein. Results of this study are indicative of ongoing transmission throughout the lockdown period, rather than re-introduction of novel lineages past lockdown. The use of molecular epidemiology in Italy should be promoted in order to provide additional understanding of the transmission of the disease and to have major effect on controlling the spread of disease.

Bloodstream infections caused by Escherichia coli carrying mcr-1 gene in hospitalized patients in northern Italy from 2012 to 2018.

PURPOSE: The recurrence of multi-drug resistant (MDR) pathogens to the latest antibiotics and the limited development of new antibacterial agents have reduced the options for the treatment of severe infections. The reintroduction of old antibiotics, such as colistin, represents an effective strategy, since the latest antibiotics are over-consumed and ineffective against MDR pathogens. In 2015, Liu (Lancet Infect Dis 16:161-168, 2016) reported Escherichia coli (E. coli) isolates carrying plasmid-mediated colistin resistance gene mcr-1. The first of mcr-1 positive colistin-resistant (col-R) E. coli from a human blood culture was observed in 2012 in Latin America, while in Italy was reported for the first time by our center in 2016. The present study aimed to describe the prevalence of mcr-1 positive col-R strains in E. coli-related bloodstream infection among patients hospitalized in Fondazione IRCCS Policlinico San Matteo in Pavia, Italy, from 2012 to 2018, including the three cases already published. METHODS: All col-R E. coli strains isolated from blood cultures collected during the study period were analyzed. The minimal inhibitory concentration of colistin was determined using broth microdilution and detection of mcr-1 and mcr-2 genes was performed by PCR. The sequence type of E. coli mcr-1 positive was determined according to Multilocus sequence typing. RESULTS: Out of 1557 samples, 14 strains (0.90%) were col-R. and positive for the presence of the mcr-1 gene, with no mcr-2 detected. The most common ST was ST10 (n = 3), followed by ST410 (n = 2). The remaining strains exhibited different MLST profiles, indicating that they were genetically unrelated. CONCLUSIONS: Proper reporting of the presence of mcr-1 genes is an essential component to anticipate the spread of colistin resistance. This public health issue is particularly alarming in Italy due to the consistent circulation of MDR bacteria.

Comparative analysis of SARS-CoV-2 quasispecies in the upper and lower respiratory tract shows an ongoing evolution in the spike cleavage site.

Studies are needed to better understand the genomic evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to describe viral quasispecies population of upper and lower respiratory tract by next-generation sequencing in patients admitted to intensive care unit. A deep sequencing of the S gene of SARS-CoV-2 from 109 clinical specimens, sampled from the upper respiratory tract (URT) and lower respiratory tract (LRT) of 77 patients was performed. A higher incidence of non-synonymous mutations and indels was observed in the LRT among minority variants. This might be explained by the ability of the virus to invade cells without interacting with ACE2 (e.g. exploiting macrophage phagocytosis). Minority variants are highly concentrated around the gene portion encoding for the Spike cleavage site, with a higher incidence in the URT; four mutations are highly recurring among samples and were found associated with the URT. Interestingly, 55.8% of minority variants detected in this locus were T>G and G>T transversions. Results from this study evidenced the presence of selective pressure and suggest that an evolutionary process is still ongoing in one of the crucial sites of spike protein associated with the spillover to humans.

Genomic epidemiology of SARS-CoV-2 reveals multiple lineages and early spread of SARS-CoV-2 infections in Lombardy, Italy.

From February to April 2020, Lombardy (Italy) reported the highest numbers of SARS-CoV-2 cases worldwide. By analyzing 346 whole SARS-CoV-2 genomes, we demonstrate the presence of seven viral lineages in Lombardy, frequently sustained by local transmission chains and at least two likely to have originated in Italy. Six single nucleotide polymorphisms (five of them non-synonymous) characterized the SARS-CoV-2 sequences, none of them affecting N-glycosylation sites. The seven lineages, and the presence of local transmission clusters within three of them, revealed that sustained community transmission was underway before the first COVID-19 case had been detected in Lombardy.

Residual SARS-CoV-2 RNA in nasal swabs of convalescent COVID-19 patients: Is prolonged quarantine always justified?

Real-time reverse transcription PCR is currently the most sensitive method to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Defining whether a patient could be contagious or not contagious in the presence of residual SARS-CoV-2 RNA is of extreme importance in the context of public health. In this prospective multicenter study, virus isolation was prospectively attempted in 387 nasal swabs from clinically recovered patients showing low viral load (quantification cycle, Cq, value greater than 30). The median Cq value was 36.8 (range 30.0-39.4). Overall, a cytopathic effect was detected in nine samples, corresponding to a culture positivity rate of 2.3% (9/387). The results of this study help to dissect true virus replication and residual viral RNA detection in recovered patients.

Rapid and sensitive detection of SARS-CoV-2 RNA using the Simplexa™ COVID-19 direct assay.

BACKGROUND: So far, one of the major drawbacks of the available molecular assays for the diagnosis of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) is the need for viral nucleic acid extraction from clinical specimens. OBJECTIVE: The aim of this study was to evaluate the performances of a newly designed real-time RT-PCR (Simplexa™ COVID-19 Direct assay), that is established with an all-in-one reagent mix and no separate extraction required. RESULTS: The lower limit of detection (LOD) for both target genes resulted the same: 3.2 (CI: 2.9-3.8) log10 cp/mL and 0.40 (CI: 0.2-1.5) TCID50/mL for S gene while 3.2 log10 (CI: 2.9-3.7) log10 cp/mL and 0.4 (CI: 0.2-1.3) TCID50/mL for ORF1ab. The LOD obtained with extracted viral RNA for both S gene or ORF1ab was 2.7 log10 cp/mL. Crossreactive analysis performed in 20 nasopharyngeal swabs confirmed a 100% of clinical specificity of the assay. Clinical performances of Simplexa™ COVID-19 Direct assay were assessed in 278 nasopharyngeal swabs tested in parallel with Corman's method. Concordance analysis showed an "almost perfect" agreement in SARS-CoV-2 RNA detection between the two assays, being κ = 0.938; SE = 0.021; 95% CI = 0.896-0.980. CONCLUSIONS: The high sensitivity and specificity of this new assay indicate that it is promising for laboratory diagnosis, enabling highspeed detection in just over one hour, which is significantly faster than the up to five hours currently required by traditional extraction followed by amplification technologies, thus allowing prompt decision making regarding isolation of infected patients.