Genomic epidemiology of the main SARS-CoV-2 variants in Italy between summer 2020 and winter 2021.

Since the beginning of the pandemic, SARS-CoV-2 has shown a great genomic variability, resulting in the continuous emergence of new variants that has made their global monitoring and study a priority. This work aimed to study the genomic heterogeneity, the temporal origin, the rate of viral evolution and the population dynamics of the main circulating variants (20E.EU1, Alpha and Delta) in Italy, in August 2020-January 2022 period. For phylogenetic analyses, three datasets were set up, each for a different main lineage/variant circulating in Italy in that time including other Italian and International sequences of the same lineage/variant, available in GISAID sampled in the same times. The international dataset showed 26 (23% Italians, 23% singleton, 54% mixed), 40 (60% mixed, 37.5% Italians, 1 singleton) and 42 (85.7% mixed, 9.5% singleton, 4.8% Italians) clusters with at least one Italian sequence, in 20E.EU1  clade, Alpha and Delta variants, respectively. The estimation of tMRCAs in the Italian clusters (including >70% of genomes from Italy) showed that in all the lineage/variant, the earliest clusters were the largest in size and the most persistent in time and frequently mixed. Isolates from the major Italian Islands tended to segregate in clusters more frequently than those from other part of Italy. The study of infection dynamics showed a positive correlation between the trend in the effective number of infections estimated by BSP model and the Re curves estimated by birth-death skyline plot. The present work highlighted different evolutionary dynamics of studied lineages with high concordance between epidemiological parameters estimation and phylodynamic trends suggesting that the mechanism of replacement of the SARS-CoV-2 variants must be related to a complex of factors involving the transmissibility, as well as the implementation of control measures, and the level of cross-immunization within the population.

Visceral fat inflammation and fat embolism are associated with lung's lipidic hyaline membranes in subjects with COVID-19.

BACKGROUND: Preliminary data suggested that fat embolism could explain the importance of visceral obesity as a critical determinant of coronavirus disease-2019 (COVID-19). METHODS: We performed a comprehensive histomorphologic analysis of autoptic visceral adipose tissue (VAT), lungs and livers of 19 subjects with COVID-19 (COVID-19+), and 23 people without COVID-19 (controls). Human adipocytes (hMADS) infected with SARS-CoV-2 were also studied. RESULTS: Although there were no between-group differences in body-mass-index and adipocytes size, a higher prevalence of CD68+ macrophages among COVID-19+ VAT was detected (p = 0.005) and accompanied by crown-like structures presence, signs of adipocytes stress and death. Consistently, human adipocytes were successfully infected by SARS-CoV-2 in vitro and displayed lower cell viability. Being VAT inflammation associated with lipids spill-over from dead adipocytes, we studied lipids distribution by ORO. Lipids were observed within lungs and livers interstitial spaces, macrophages, endothelial cells, and vessels lumen, features suggestive of fat embolism syndrome, more prevalent among COVID-19+ (p < 0.001). Notably, signs of fat embolism were more prevalent among people with obesity (p = 0.03) independently of COVID-19 diagnosis, suggesting that such condition may be an obesity complication exacerbated by SARS-CoV-2 infection. Importantly, all infected subjects' lungs presented lipids-rich (ORO+) hyaline membranes, formations associated with COVID-19-related pneumonia, present only in one control patient with non-COVID-19-related pneumonia. Importantly, transition aspects between embolic fat and hyaline membranes were also observed. CONCLUSIONS: This study confirms the lung fat embolism in COVID-19+ patients and describes for the first time novel COVID-19-related features possibly underlying the unfavorable prognosis in people with COVID-19 and obesity.

Circulating SARS-CoV-2 variants in Italy, October 2020-March 2021.

A growing number of emerging SARS-CoV-2 variants is being identified worldwide, potentially impacting the effectiveness of current vaccines. We report the data obtained in several Italian regions involved in the SARS-CoV-2 variant monitoring from the beginning of the epidemic and spanning the period from October 2020 to March 2021.

Quantification of the HIV-1 total reservoir in the peripheral blood of naïve and treated patients by a standardised method derived from a commercial HIV-1 RNA quantification assay.

OBJECTIVES: HIV-1 DNA can persist in host cells, establishing a latent reservoir. This study was aimed to develop an extraction and amplification protocol for HIV-1 DNA quantification by modifying a quantitative commercial assay. METHODS: HIV-1 DNA was extracted on an Abbott m2000sp instrument, using an open-mode protocol. Two calibrators, spiked with a plasmid containing HIV-1 genome (103 and 105 cps/mL), were extracted and amplified to generate a master calibration curve. Precision, accuracy, linear dynamic range, limit of quantification (LOQ) and limit of detection (LOD) were determined. A cohort of patients, naïve or chronically infected, was analysed. RESULTS: Calibration curve was obtained from 42 replicates of standards (stds); precision was calculated (coefficients of variability [CVs] below 10%); accuracy was higher than 90%. Linearity covered the entire range tested (10-104 copies per reaction), and LOD (95%) was 12 copies per reaction. HIV-1 DNA was significantly higher (p < 0.0001) in drug-naïve (62) than in chronically treated patients (50), and proviral loads correlated with lymphocytes (p = 0.0002) and CD4+ (p < 0.0001) counts only in naïve patients. Both groups displayed a significant inverse correlation between CD4+ nadir and proviral loads. A significant correlation (p < 0.0001) between viraemia and HIV-1 reservoir was disclosed. No significant difference was obtained from the comparison between proviral loads on whole blood and peripheral blood mononuclear cells (PBMCs) from the same patient. CONCLUSIONS: The novelty of our approach relies on the selection of appropriate reference standard extracted and amplified as clinical specimens avoiding any underestimation of the reservoir. Results confirm HIV-1 DNA as a marker of disease progression, supporting the relationship between the width of latent reservoir and the immunological status of the patient.

SARS-CoV-2 multi-variant rapid detector based on graphene transistor functionalized with an engineered dimeric ACE2 receptor.

Reliable point-of-care (POC) rapid tests are crucial to detect infection and contain the spread of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The emergence of several variants of concern (VOC) can reduce binding affinity to diagnostic antibodies, limiting the efficacy of the currently adopted tests, while showing unaltered or increased affinity for the host receptor, angiotensin converting enzyme 2 (ACE2). We present a graphene field-effect transistor (gFET) biosensor design, which exploits the Spike-ACE2 interaction, the crucial step for SARS-CoV-2 infection. Extensive computational analyses show that a chimeric ACE2-Fragment crystallizable (ACE2-Fc) construct mimics the native receptor dimeric conformation. ACE2-Fc functionalized gFET allows in vitro detection of the trimeric Spike protein, outperforming functionalization with a diagnostic antibody or with the soluble ACE2 portion, resulting in a sensitivity of 20 pg/mL. Our miniaturized POC biosensor successfully detects B.1.610 (pre-VOC), Alpha, Beta, Gamma, Delta, Omicron (i.e., BA.1, BA.2, BA.4, BA.5, BA.2.75 and BQ.1) variants in isolated viruses and patient's clinical nasopharyngeal swabs. The biosensor reached a Limit Of Detection (LOD) of 65 cps/mL in swab specimens of Omicron BA.5. Our approach paves the way for a new and reusable class of highly sensitive, rapid and variant-robust SARS-CoV-2 detection systems.

Phylogeography and genomic epidemiology of SARS-CoV-2 in Italy and Europe with newly characterized Italian genomes between February-June 2020.

The aims of this study were to characterize new SARS-CoV-2 genomes sampled all over Italy and to reconstruct the origin and the evolutionary dynamics in Italy and Europe between February and June 2020. The cluster analysis showed only small clusters including < 80 Italian isolates, while most of the Italian strains were intermixed in the whole tree. Pure Italian clusters were observed mainly after the lockdown and distancing measures were adopted. Lineage B and B.1 spread between late January and early February 2020, from China to Veneto and Lombardy, respectively. Lineage B.1.1 (20B) most probably evolved within Italy and spread from central to south Italian regions, and to European countries. The lineage B.1.1.1 (20D) developed most probably in other European countries entering Italy only in the second half of March and remained localized in Piedmont until June 2020. In conclusion, within the limitations of phylogeographical reconstruction, the estimated ancestral scenario suggests an important role of China and Italy in the widespread diffusion of the D614G variant in Europe in the early phase of the pandemic and more dispersed exchanges involving several European countries from the second half of March 2020.

Epidemiological and Clinical Features of SARS-CoV-2 Variants Circulating between April-December 2021 in Italy.

SARS-CoV-2 is constantly evolving, leading to new variants. We analysed data from 4400 SARS-CoV-2-positive samples in order to pursue epidemiological variant surveillance and to evaluate their impact on public health in Italy in the period of April-December 2021. The main circulating strain (76.2%) was the Delta variant, followed by the Alpha (13.3%), the Omicron (5.3%), and the Gamma variants (2.9%). The B.1.1 lineages, Eta, Beta, Iota, Mu, and Kappa variants, represented around 1% of cases. There were 48.2% of subjects who had not been vaccinated, and they had a lower median age compared to the vaccinated subjects (47 vs. 61 years). An increasing number of infections in the vaccinated subjects were observed over time, with the highest proportion in November (85.2%). The variants correlated with clinical status; the largest proportion of symptomatic patients (59.6%) was observed with the Delta variant, while subjects harbouring the Gamma variant showed the highest proportion of asymptomatic infection (21.6%), albeit also deaths (5.4%). The Omicron variant was only found in the vaccinated subjects, of which 47% had been hospitalised. The diffusivity and pathogenicity associated with the different SARS-CoV-2 variants are likely to have relevant public health implications, both at the national and international levels. Our study provides data on the rapid changes in the epidemiological landscape of the SARS-CoV-2 variants in Italy.

High prevalence of carbapenem-resistant Klebsiella pneumoniae ST307 recovered from fecal samples in an Italian hospital.

Aim: This study reports the characterization of carbapenem-resistant colonizing strains of K. pneumoniae. Methods: 650 stool samples were screened for carbapenem-resistant K. pneumoniae (CR-Kp). All strains were characterized for antibiotic susceptibility, typing features, main carbapenemases and extended-spectrum ß-lactamases. The carbapenemase transferability was assessed by interspecific conjugation. Results: Eighteen CR-Kp were multidrug resistant, five were KPC producing. A predominance of ST307 isolates, constituting the predominant cluster by PFGE analysis, was identified (50% were KPC-2 producers). Conjugation data showed the co-transfer of blaKPC-2, blaTEM-1, blaOXA-1, blaCTX-M-15 in a single large pKPN3-like plasmid. Conclusion: Our data pointed out the diversity of colonizing K. pneumoniae strains compared with clinical ones. The predominance of ST307 strains suggested an increased spreading, even in our area, of this high-risk clone.

Lay abstract Carbapenem-resistant Klebsiella pneumoniae represents a major antibiotic resistance threat worldwide. These microorganisms are associated with high mortality and difficult-to-treat infections. Of particular interest is the production of carbapenemase, enzymes capable of inactivating life-saving drugs such as carbapenems. In the interaction with humans, K. pneumoniae plays different roles: commensal, opportunistic pathogen or true pathogen. Our study aimed to analyze the population of K. pneumoniae obtained from a fecal screening, since gut-colonizing strains are considered the common source of K. pneumoniae nosocomial infections. There are many differences between clinical and colonizing isolates, but the latter are much less characterized. The careful characterization of colonizing strains is crucial, in order to better understand how K. pneumoniae may change its role from commensal to pathogen.

Weak Cross-Lineage Neutralization by Anti SARS-CoV-2 Spike Antibodies after Natural Infection or Vaccination Is Rescued by Repeated Immunological Stimulation.

After over one year of evolution, through billions of infections in humans, SARS-CoV-2 has evolved into a score of slightly divergent lineages. A few different amino acids in the spike proteins of these lineages can hamper both natural immunity against reinfection, and vaccine efficacy. In this study, the in vitro neutralizing potency of sera from convalescent COVID-19 patients and vaccinated subjects was analyzed against six different SARS-CoV-2 lineages, including the latest B.1.617.2 (or Delta variant), in order to assess the cross-neutralization by anti-spike antibodies. After both single dose vaccination, or natural infection, the neutralizing activity was low and fully effective only against the original lineage, while a double dose or a single dose of vaccine, even one year after natural infection, boosted the cross-neutralizing activity against different lineages. Neither binding, nor the neutralizing activity of sera after vaccination, could predict vaccine failure, underlining the need for additional immunological markers. This study points at the importance of the anamnestic response and repeated vaccine stimulations to elicit a reasonable cross-lineage neutralizing antibody response.

Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing.

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.

Trend of clinical vancomycin-resistant enterococci isolated in a regional Italian hospital from 2001 to 2018.

A retrospective study of the epidemiology of vancomycin-resistant enterococci (VRE) in a regional hospital of central Italy in 2001-2018 demonstrated an increased VRE prevalence since 2016. A total of 113 VRE isolates, 89 E. faecium (VREfm) and 24 E. faecalis (VREfs), were collected in the study period. All strains showed high-level resistance to vancomycin; 107 also showed teicoplanin resistance. Altogether, 84 VREfm and 20 VREfs carried vanA, whereas 5 VREfm and 1 VREfs carried vanB. MLST analysis documented that 89 VREfm isolates mainly belonged to ST78, ST80, and ST117. Most strains were isolated from 2001 to 2007, ST78 being the predominant clone. VREfm re-emerged in 2016 with a prevalence of the ST80 lineage. Most VREfs were isolated from 2001 to 2006; although they belonged to 7 different STs, there was a prevalence of ST88 and ST6. Notably, ST88 was sporadically recovered throughout the study period. The increasing rate of VREfm isolation from 2016 to 2018 may be related to the influx of new successful clones and to the renewed and widespread use of vancomycin. Improved infection control measures in hospital wards should be adopted to limit the spread of new epidemic VRE strains.

Molecular Tracing of SARS-CoV-2 in Italy in the First Three Months of the Epidemic.

The aim of this study is the characterization and genomic tracing by phylogenetic analyses of 59 new SARS-CoV-2 Italian isolates obtained from patients attending clinical centres in North and Central Italy until the end of April 2020. All but one of the newly-characterized genomes belonged to the lineage B.1, the most frequently identified in European countries, including Italy. Only a single sequence was found to belong to lineage B. A mean of 6 nucleotide substitutions per viral genome was observed, without significant differences between synonymous and non-synonymous mutations, indicating genetic drift as a major source for virus evolution. tMRCA estimation confirmed the probable origin of the epidemic between the end of January and the beginning of February with a rapid increase in the number of infections between the end of February and mid-March. Since early February, an effective reproduction number (Re) greater than 1 was estimated, which then increased reaching the peak of 2.3 in early March, confirming the circulation of the virus before the first COVID-19 cases were documented. Continuous use of state-of-the-art methods for molecular surveillance is warranted to trace virus circulation and evolution and inform effective prevention and containment of future SARS-CoV-2 outbreaks.

Increase and diversity of carbapenemase-producing Escherichia coli isolates, Italy.

Aim: This study reports on a surveillance in an Italian hospital focused on carbapenemase-producing Escherichia coli (CP-Ec). Materials & methods: Eighteen isolates (nine from clinical specimens and nine from rectal swab) were characterized for antibiotic susceptibilities, typing features, main carbapenemase, extended-spectrum ß-lactamases (ESBLs) and other bla genes, and their transferability by conjugation and transformation. Results: An increase in CP-Ec isolates was observed during 3-year surveillance period. Compared with the clinical isolates, all belonging to one sequence type (ST), ST131, those from rectal swab were very heterogeneous and belonged to eight STs. Transfer data confirmed the role of conjugative plasmids in the spreading of carbapenemase genes. Conclusion: The prevalence of CP-Ec in Italy has risen, with a substantial increase over the last year.