A Cholera Case Imported from Bangladesh to Italy: Clinico-Epidemiological Management and Molecular Characterization in a Non-Endemic Country.

Despite the number of cholera outbreaks reported worldwide, only a few cases are recorded among returning European travellers. We describe the case of a 41-year-old male, returning to Italy after a stay in Bangladesh, his origin country, who presented with watery diarrhoea. Vibrio cholerae and norovirus were detected in the patient's stools via multiplex PCR methods. Direct microscopy, Gram staining, culture and antibiotic susceptibility tests were performed. The isolates were tested using end-point PCR for the detection of potentially enteropathogenic V. cholera. Serotype and cholera toxins identification were carried out. Whole genome sequencing and bioinformatics analysis were performed, and antimicrobial resistance genes identified. A phylogenetic tree with the most similar genomes of databases previously described was built. Sample of the food brought back by the patient were also collected and analysed. The patient was diagnosed with V. cholerae O1, serotype Inaba, norovirus and SARS-CoV-2 concomitant infection. The isolated V. cholerae strain was found to belong to ST69, encoding for cholera toxin, ctxB7 type and was phylogenetically related to the 2018 outbreak in Dhaka, Bangladesh. Adopting a multidisciplinary approach in a cholera non-endemic country ensured rapid and accurate diagnosis, timely clinical management, and epidemiological investigation at national and international level.

Molecular Characterization of Whole-Genome SARS-CoV-2 from the First Suspected Cases of the XE Variant in the Lazio Region, Italy.

We report two cases of SARS-CoV-2 recombinant variant XE detected in nasopharyngeal swabs (NPS) of hospitalized patients with no evident epidemiological link in Lazio, Central Italy. Whole-Genome Sequencing (WGS) performed on an Ion Torrent GSS5 platform according to Italian flash surveys showed genomes corresponding to the PANGOLIN unclassified lineage and the Nextclade XE clade. Further analyses were then carried out to investigate more deeply the genetic characteristics of these XE-like sequences. When phylogenetic trees, by using IQ-TREE, were built splitting the genome into two regions according to the putative XE recombination site, the upstream and downstream regions were seen to be clustered near BA.1 and BA.2 sequences, respectively. However, our XE-like sequences clustered separately, with a significant bootstrap, from the classified European and Italian XE strains, although the recombination site between BA.1 and BA.2 was identified at the nucleotide site 11556 by RDP4 software, consistent with the putative XE breakpoint. These findings show the risk of the introduction of novel recombinant variants of SARS-CoV-2 and the existence of XE-like strains, phylogenetically separated, that could make their exact taxonomy difficult. It follows the need for continued SARS-CoV-2 surveillance by WGS.

Air pollution, SARS-CoV-2 incidence and COVID-19 mortality in Rome – a longitudinal study

Chronic exposure to ambient air pollution has been related to increased mortality in the general population [1]. After the outbreak of SARS-CoV-2 pandemic in 2019, there has been a fast proliferation of epidemiological studies linking ambient air pollution to COVID-19 incidence or adverse prognosis [2]. It has been hypothesised that ambient air pollution might increase human vulnerability to viruses by reducing immune defences, promoting a low-level chronic inflammatory state, or leading to chronic diseases [3]. Most studies applied ecological designs, and failed to account for key individual-level or area-level determinants of COVID-19 spread or severity, such as demographic characteristics of the studied populations, socioeconomic or clinical susceptibility, and area-level proxies of disease spread such as mobility or population density [4].

[Mortality and impact of COVID-19 by citizenship in seven Italian Regions from the beginning of the pandemic to mid-July 2021]. / Mortalità per COVID-19 nella popolazione immigrata in sette Regioni italiane da inizio pandemia a metà luglio 2021.

OBJECTIVES: to quantify the variability of COVID-19 mortality from the beginning of the pandemic to mid-July 2021, in relation to the immigrant status and by Region and period. DESIGN: observational incidence study. SETTING AND PARTICIPANTS: the study population consists of the residents at the beginning of 2020 in seven Regions (Piedmont, Lombardy, Veneto, Emilia-Romagna, Tuscany, Lazio, Sicily) aged <=74 years. MAIN OUTCOME MEASURES: absolute frequency of deaths occurred in subjects who tested positive for SARS-CoV-2, crude and standardized rates (standard: Italian population at the beginning of 2020), and mortality rates ratios (obtained using Poisson models), by immigrant status and stratified by gender, Region of residence, and period. The study period was divided into 5 subperiods: 22.02.2020-25.05.2020, 26.05.2020-02.10.2020, 03.10.2020-26.02.2021, 27.02.2021-16.07.2021. RESULTS: the study includes more than one half of the Italian population and most of the immigrants residing in the country, who are younger than Italians and experienced fewer COVID-19 deaths. Deaths among those who tested positive varied greatly between Regions and periods; standardized rates showed considerable increases over time among immigrants. In terms of rate ratios, there were excesses among immigrant males in the third period (MRR: 1.46; 95%CI 1.30-1.65) and in the fourth period (MRR: 1.55; 95%CI 1, 34-1.81). Among immigrant females, there is an indication of lower risk in the third period (MRR: 0.79; 95%CI 0.65-0.97) and of greater risk in the fourth period (MRR: 1. 46; 95%CI 1.21-1.77). Finally, the effect is modified by the Region of residence, both in the third and in the fourth period for males and only in the fourth period for females. CONCLUSIONS: the risk of premature mortality due to COVID-19 is linked to immigrant status and with an intensity that varies by gender, Region, and period. More accessible tools for prevention, diagnosis and early healthcare can support immigrant communities in managing the risk factors linked to the spread of infections and, in particular, counteract their evolution into more severe disease outcomes.

[Impact of the COVID-19 epidemic on total and cause-specific mortality in Rome (Italy) in 2020]. / Impatto dell'epidemia di COVID-19 sulla mortalità totale e per causa a Roma nel 2020.

OBJECTIVES: to estimate the impact of the COVID-19 epidemic on total and cause-specific mortality in people residing and dead in the Municipality of Rome (Italy) in 2020, and to describe the causes of death of subjects with SARS-CoV-2 infection confirmed by molecular test. DESIGN: descriptive analysis of total and cause-specific mortality in 2020 in Rome and comparison with a reference period (2015-2018 for total mortality and 2018 for cause-specific mortality); descriptive analysis of cause-specific mortality in the cohort of SARS-CoV-2 infected subjects. SETTING AND PARTICIPANTS: 27,471 deaths registered in the Lazio mortality-cause Registry, relating to people residing and died in the municipality of Rome in 2020, 2,374 of which died from COVID-19.MAIN OUCOME MEASURES: all-cause mortality by month, gender, age group and place of death, cause-specific mortality (ICD-10 codes). RESULTS: in the municipality of Rome in 2020, an excess of mortality from all causes equal to +10% was observed, with a greater increase in the months of October-December (+27%, +56%, and +26%, respectively) in people aged 50+, with the greatest contribution from the oldest age groups (80+) who died in the nursing homes or at home. Lower mortality was observed in the age groups 0-29 years (-30%) and 40-49 years (-13%). In 2020, COVID-19 represents the fourth cause of death in Rome after malignant tumours, diseases of the circulatory system, and respiratory diseases. Excess mortality was observed from stroke and pneumonia (both in men and women), from respiratory diseases (in men), from diabetes, mental disorders, dementia and Parkinson's disease (in women). On the contrary, mortality is lower for all cancers, for diseases of the blood and haematopoietic organs and for the causes of the circulatory system. The follow-up analysis of SARS-CoV-2 positive subjects residing in Rome shows that a share of deaths (about 20%) reports other causes of death such as cardiovascular diseases, malignant tumours, and diseases of the respiratory system on the certificate collected by the Italian National Statistics Institute. CONCLUSIONS: the 2020 mortality study highlighted excesses for acute and chronic pathologies, indicative of possible delays in the diagnosis or treatment of conditions indirectly caused by the pandemic, but also a share of misclassification of the cause of death that is recognized as COVID-19 death.

COVID-19 vaccines coverage and effectiveness against SARS-CoV-2 infection among residents in the largest Health Authority of Lazio region (Italy): a population-based cohort study.

BACKGROUND: The waning of the protective effect of COVID-19 vaccines and timing of booster doses are debated. METHODS: Population-based cohort study in the largest Health-Authority of Lazio region, Italy, on 946,156 residents aged 12+ (study period: 1 January 2021-10 January 2022). Vaccine effectiveness (VE) against any SARS-CoV-2 infection (symptomatic or asymptomatic) was estimated through multivariable negative-binomial models using unvaccinated person-time as a reference. RESULTS: The primary vaccination cycle was completed by 81% of residents; of these, 45% received a booster dose. Vaccine coverages were lower for foreigners, and people living in deprived areas, families with children aged 0-11, and households size 1 or 6+. Overall, VE waned from 71% (95% Confidence Interval (CI) 70-73%) 1 month after the second dose to 43% (CI 41-45%) after 4 months and 24% (CI 21-27%) after 6 months, especially in the elderly aged 70+. We observed a prompt restore of VE 15-19 days after the booster dose (69%, CI 67-70%). CONCLUSIONS: Our results support the recommendation of a booster dose 4 months after completion of the primary cycle, giving priority to elderly and fragile individuals. The lower vaccine coverage among social disadvantaged subgroups suggests the need of targeted communication and interventions.

Virological and Serological Characterisation of SARS-CoV-2 Infections Diagnosed After mRNA BNT162b2 Vaccination Between December 2020 and March 2021.

BACKGROUND: Vaccines for coronavirus disease 2019 (COVID-19) are proving to be very effective in preventing severe illness; however, although rare, post-vaccine infections have been reported. The present study focuses on virological and serological features of 94 infections that occurred in Lazio Region (Central Italy) between 27 December 2020, and 30 March 2021, after one or two doses of mRNA BNT162b2 vaccine. METHODS: We evaluated clinical features, virological (viral load; viral infectiousness; genomic characterisation), and serological (anti-nucleoprotein Ig; anti-Spike RBD IgG; neutralising antibodies, nAb) characteristics of 94 post-vaccine infections at the time of diagnosis. Nasopharyngeal swabs (NPSs) and serum samples were collected in the framework of the surveillance activities on SARS-CoV-2 variants established in Lazio Region (Central Italy) and analysed at the National Institute for Infectious Diseases "L. Spallanzani" in Rome. RESULTS: The majority (92.6%) of the post-vaccine infections showed pauci/asymptomatic or mild clinical course, with symptoms and hospitalisation rate significantly less frequent in patients infected after full vaccination course as compared to patients who received a single dose vaccine. Although differences were not statistically significant, viral loads and isolation rates were lower in NPSs from patients infected after receiving two vaccine doses as compared to patients with one dose. Most cases (84%) had nAb in serum at the time of infection diagnosis, which is a sub-group of vaccinees, were found similarly able to neutralise Alpha and Gamma variants. Asymptomatic individuals showed higher nAb titres as compared to symptomatic cases (median titre: 1:120 vs. 1:40, respectively). Finally, the proportion of post-vaccine infections attributed either to Alpha and Gamma variants was similar to the proportion observed in the contemporary unvaccinated population in the Lazio region, and mutational analysis did not reveal enrichment of a defined set of Spike protein substitutions depending on the vaccination status. CONCLUSION: Our study conducted using real-life data, emphasised the importance of monitoring vaccine breakthrough infections, through the characterisation of virological, immunological, and clinical features associated with these events, in order to tune prevention measures in the next phase of the COVID-19 pandemic.

COVID-19 Rapid Antigen Test as Screening Strategy at Points of Entry: Experience in Lazio Region, Central Italy, August-October 2020.

COVID-19 pandemic is a dramatic health, social and economic global challenge. There is urgent need to maximize testing capacity. Rapid Antigen Tests (RAT) represent good candidates for point-of-care and mass surveillance testing to rapidly identify SARS-CoV-2-infected people, counterbalancing lower sensitivity vs. gold standard molecular tests with fast results and possible recurrent testing. We describe the results obtained with the testing algorithm implemented at points of entry (airports and ports) in the Lazio Region (Italy), using the STANDARD F COVID-19 Antigen Fluorescence ImmunoAssay (FIA), followed by molecular confirmation of FIA-positive samples. From mid-August to mid-October 2020, 73,643 RAT were reported to the Regional Surveillance Information System for travelers at points of entry in Lazio Region. Of these, 1176 (1.6%) were FIA-positive, and the proportion of RT-PCR-confirmed samples was 40.5%. Our data show that the probability of confirmation was directly dependent from the semi-quantitative FIA results. In addition, the molecularly confirmed samples were those with high levels of virus and that were actually harboring infectious virus. These results support public health strategies based on early mass screening campaigns by RAT in settings where molecular testing is not feasible or easily accessible, such as points of entry. This approach would contribute to promptly controlling viral spread through travel, which is now of particular concern due to the spread of SARS-CoV-2 variants.

SARS-CoV-2 Early Screening at the Point of Entry: Travelers From Bangladesh to Italy-July 2020.

We report phylogenetic and mutational analysis by NGS of six SARS-CoV-2 strains from patients flying from Bangladesh to Italy (July 2020). Data suggest that no further circulation of such imported strains occurred in Italy, stating the efficacy of early screening at the point of entry and supporting the importance of molecular epidemiology in monitoring the efficacy of control measures.

[Spatial analysis for detecting clusters of cases during the COVID-19 emergency in Rome and in the Lazio Region (Central Italy)]. / Analisi spaziale per l'identificazione di cluster di casi durante l'emergenza COVID-19 a Roma e nel Lazio.

BACKGROUND: one of the most affected European countries by the COVID-19 epidemic is Italy; data show the strong geographical heterogeneity of the epidemic. OBJECTIVES: to propose an analysis strategy to ascertain the non-random nature of the spatial spread of COVID-19 cases infection and identify any territorial aggregations, in order to enhance contact tracing activities in specific areas of the Lazio Region (Central Italy) and a large urban area as Rome. METHODS: all cases of COVID-19 of the Lazio Region notified to the Regional Service for Epidemiology, Surveillance, and Control of Infectious Diseases (Seresmi) with daily updates from the beginning of the epidemic to April 27, 2020 were considered. The analyses were carried out considering two periods (the first from the beginning of the epidemic to April 6 and the second from the beginning of the epidemic to April 27) and two different levels of spatial aggregation: the entire Lazio region excluding the Municipality of Rome, where the 377 municipalities represent the area units, and the Municipality of Rome, where the area units under study are the 155 urban areas (ZUR). The Scan statistic of Kulldorff was used to ascertain the non-random nature of the spatial spread of infected cases and to identify any territorial aggregations of cases of COVID-19 infection, using a retrospective spatial analysis in two overlapping periods. RESULTS: analysis was conducted at regional level in the two survey periods and revealed the presence of 7 localized clusters. In the Municipality of Rome, a single cluster (Historic Centre) was identified in the first period which includes 7 urban areas, while in the second period two distinct clusters (Omo and Farnesina) were observed. CONCLUSIONS: Scan statistics are an important surveillance tool for monitoring disease outbreaks during the active phase of the epidemic and a useful contribution to epidemiological surveillance during the COVID-19 epidemic in a specific territory.

COVID-19 disease-Temporal analyses of complete blood count parameters over course of illness, and relationship to patient demographics and management outcomes in survivors and non-survivors: A longitudinal descriptive cohort study.

BACKGROUND: Detailed temporal analyses of complete (full) blood count (CBC) parameters, their evolution and relationship to patient age, gender, co-morbidities and management outcomes in survivors and non-survivors with COVID-19 disease, could identify prognostic clinical biomarkers. METHODS: From 29 January 2020 until 28 March 2020, we performed a longitudinal cohort study of COVID-19 inpatients at the Italian National Institute for Infectious Diseases, Rome, Italy. 9 CBC parameters were studied as continuous variables [neutrophils, lymphocytes, monocytes, platelets, mean platelet volume, red blood cell count, haemoglobin concentration, mean red blood cell volume and red blood cell distribution width (RDW %)]. Model-based punctual estimates, as average of all patients' values, and differences between survivors and non-survivors, overall, and by co-morbidities, at specific times after symptoms, with relative 95% CI and P-values, were obtained by marginal prediction and ANOVA- style joint tests. All analyses were carried out by STATA 15 statistical package. MAIN FINDINGS: 379 COVID-19 patients [273 (72% were male; mean age was 61.67 (SD 15.60)] were enrolled and 1,805 measures per parameter were analysed. Neutrophils' counts were on average significantly higher in non-survivors than in survivors (P<0.001) and lymphocytes were on average higher in survivors (P<0.001). These differences were time dependent. Average platelets' counts (P<0.001) and median platelets' volume (P<0.001) were significantly different in survivors and non-survivors. The differences were time dependent and consistent with acute inflammation followed either by recovery or by death. Anaemia with anisocytosis was observed in the later phase of COVID-19 disease in non-survivors only. Mortality was significantly higher in patients with diabetes (OR = 3.28; 95%CI 1.51-7.13; p = 0.005), obesity (OR = 3.89; 95%CI 1.51-10.04; p = 0.010), chronic renal failure (OR = 9.23; 95%CI 3.49-24.36; p = 0.001), COPD (OR = 2.47; 95% IC 1.13-5.43; p = 0.033), cardiovascular diseases (OR = 4.46; 95%CI 2.25-8.86; p = 0.001), and those >60 years (OR = 4.21; 95%CI 1.82-9.77; p = 0.001). Age (OR = 2.59; 95%CI 1.04-6.45; p = 0.042), obesity (OR = 5.13; 95%CI 1.81-14.50; p = 0.002), renal chronic failure (OR = 5.20; 95%CI 1.80-14.97; p = 0.002) and cardiovascular diseases (OR 2.79; 95%CI 1.29-6.03; p = 0.009) were independently associated with poor clinical outcome at 30 days after symptoms' onset. INTERPRETATION: Increased neutrophil counts, reduced lymphocyte counts, increased median platelet volume and anaemia with anisocytosis, are poor prognostic indicators for COVID19, after adjusting for the confounding effect of obesity, chronic renal failure, COPD, cardiovascular diseases and age >60 years.

Performance evaluation of Abbott ARCHITECT SARS-CoV-2 IgG immunoassay in comparison with indirect immunofluorescence and virus microneutralization test.

BACKGROUND: Serological tests for anti-SARS-CoV-2 antibodies are becoming of great interest to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. OBJECTIVES: We evaluated the diagnostic performance of the Abbott ARCHITECT SARS-CoV-2 IgG test, a fully automated indirect immunoassay that detects antibodies directed to a recombinant SARS-CoV-2 Nucleocapsid antigen. STUDY DESIGN: Abbott ARCHITECT SARS-CoV-2 IgG immunoassay was compared to an indirect immunofluorescence assay (IFA) on sera from patients with COVID-19 collected at different days after symptoms onset or infected by other human coronaviruses. Comparison with neutralization test was also performed. RESULTS: After 7, 14 and >14 days after onset ARCHITECT was positive on 8.3 %; 61.9 % and 100 % of the tested samples compared to 58.3 %; 85.7 % and 100 % by IFA. The sensitivity was 72 % vs. IFA and 66.7 % vs. a real-time PCR, the specificity was 100 %. On 18 samples with neutralizing activity, 17 were positive by Abbott ARCHITECT SARS-CoV-2 IgG. CONCLUSIONS: In our study, Abbott ARCHITECT SARS-CoV-2 IgG assay showed a satisfactory performance, with a very high specificity. IgG reactivity against SARSCoV-2 N antigen was detectable in all patients by two weeks after symptoms onset. In addition, concordance between this serological response and viral neutralization suggests that a strong humoral response may be predictive of a neutralization activity, regardless of the target antigens. This finding supports the use of this automated serological assay in diagnostic algorithm and public health intervention, especially for high loads of testing.

COVID-19: viral-host interactome analyzed by network based-approach model to study pathogenesis of SARS-CoV-2 infection.

BACKGROUND: Epidemiological, virological and pathogenetic characteristics of SARS-CoV-2 infection are under evaluation. A better understanding of the pathophysiology associated with COVID-19 is crucial to improve treatment modalities and to develop effective prevention strategies. Transcriptomic and proteomic data on the host response against SARS-CoV-2 still have anecdotic character; currently available data from other coronavirus infections are therefore a key source of information. METHODS: We investigated selected molecular aspects of three human coronavirus (HCoV) infections, namely SARS-CoV, MERS-CoV and HCoV-229E, through a network based-approach. A functional analysis of HCoV-host interactome was carried out in order to provide a theoretic host-pathogen interaction model for HCoV infections and in order to translate the results in prediction for SARS-CoV-2 pathogenesis. The 3D model of S-glycoprotein of SARS-CoV-2 was compared to the structure of the corresponding SARS-CoV, HCoV-229E and MERS-CoV S-glycoprotein. SARS-CoV, MERS-CoV, HCoV-229E and the host interactome were inferred through published protein-protein interactions (PPI) as well as gene co-expression, triggered by HCoV S-glycoprotein in host cells. RESULTS: Although the amino acid sequences of the S-glycoprotein were found to be different between the various HCoV, the structures showed high similarity, but the best 3D structural overlap shared by SARS-CoV and SARS-CoV-2, consistent with the shared ACE2 predicted receptor. The host interactome, linked to the S-glycoprotein of SARS-CoV and MERS-CoV, mainly highlighted innate immunity pathway components, such as Toll Like receptors, cytokines and chemokines. CONCLUSIONS: In this paper, we developed a network-based model with the aim to define molecular aspects of pathogenic phenotypes in HCoV infections. The resulting pattern may facilitate the process of structure-guided pharmaceutical and diagnostic research with the prospect to identify potential new biological targets.

SARS-CoV-2 Phylogenetic Analysis, Lazio Region, Italy, February-March 2020.

We report phylogenetic and mutational analysis of severe acute respiratory syndrome coronavirus 2 virus strains from the Lazio region of Italy and provide information about the dynamics of virus spread. Data suggest effective containment of clade V strains, but subsequently, multiple waves of clade G strains were circulating widely in Europe.

Coronavirus disease (COVID-19) in a paucisymptomatic patient: epidemiological and clinical challenge in settings with limited community transmission, Italy, February 2020.

Data concerning the transmission of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) in paucisymptomatic patients are lacking. We report an Italian paucisymptomatic case of coronavirus disease 2019 with multiple biological samples positive for SARS-CoV-2. This case was detected using the World Health Organization protocol on cases and contact investigation. Current discharge criteria and the impact of extra-pulmonary SARS-CoV-2 samples are discussed.