SARS Cov2 experience in the south of Italy

Coronavirus 2 (CoV2) is challenging health care and economical asset all over the world. We report our experience in the CoV2 unit in a southern, Italian community hospital. Eighty-nine patients came to our observation for suspected CoV2 infection. After clinical evaluation, oro-pharyngeal and/or nasal swabs, blood withdrawals and radiological examinations were performed. At admission, Brescia Covid Scale was higher in non-survivors compared to survivors, especially in lymphopenic patients. Higher levels of C Reactive Protein (CRP) and Procalcitonin (PCT) were present in lymphopenic, non-survivors compared to survivors. Creatinine (Cre) was higher both in lymphopenic and no lymphopenic non-survivors compared to survivors. At the last control, a decrease in CRP in all survivors, an increase of PCT in all non-survivors were revealed. A lower absolute lymphocyte count and higher d-dimer and creatinine were present in lymphopenic, non-survivors. CRP and creatinine increased, while absolute lymphocyte count decreased in no lymphopenic, non-survivors. The majority of our patients had interstitial pneumonia with typical ground-glass lesions. Comorbidities weighted on disease evolution. Overall the mortality was low compared to the northern of Italy, because of early diagnosis and therapy, screening on all the inhabitants, low level of environmental pollution. Our data highlight the pivotal role of classifying severity of clinical conditions according to the comorbidities and the immunological asset. We propose a scale for promptly defining clinical setting, treatments and prognosis. *Correspondence to: Fiori Patrizia, Neurological Unit, S. Ottone Frangipane Hospital, Ariano irpino (AV), ASL AV, University of Naples, Italy, Tel: 39-825877602; Fax 39-825-828409; E-mail patriziafiorirmit@alice.it


Introduction
Coronaviruses (CoV) were described as mite agents, causing benign, self-limiting upper respiratory tract and intestinal infections. Asian outbreaks of CoV-related Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) pointed out the risk of contagion diffusion, related morbidity and mortality (the latter being around 10% and 35%, respectively) [1][2][3][4][5]. Pandemia of CoV2 SARS threatened health care and economical steady state all over the world. This virus is homologous with human SARS and MERS viruses [6]. It is a small, round-oval, single, positive-strand RNA b-CoV (Sarbecovirus subgenus, Orthocoronavirinae subfamily, Nidovirales order), of 60-140 nm in diameter. It is constituted by six essential structural proteins, namely spike (S) glycoprotein, small envelope (E) protein, matrix (M) protein, nucleocapsid (N) protein, a replicase transcriptase complex (RTC), with two overlapping open reading frames (ORFa and ORFb).
The name of CoV was conferred because of peculiar crown aspect of club-shape S projections on the membrane. S1 protein anchors on host Angiotensin Converting Enzyme 2 Receptor (Ace2R) and S2 protein mediates viral-cell membrane fusion by two tandem domains, heptad repeats 1 and 2. Transmembrane Serine Protease 2, phosphoinositide 5 kinase, 2-pore-segment channel 2 and cathepsin L play a critical role in viral penetration into the cells. By viral RNA translation into the cytoplasm, the RTC is activated, transcription is started up, viral synthesis, replication and release rapidly occur [7][8][9][10][11][12][13]. M protein is a structural protein, determining viral shape. Interactions of N protein tethers viral RNA to RTC and packages the encapsidated genome into viral particles. E is a transmembrane protein with ion channel activity, facilitating viral assembly and release. Exceeding S proteins, not assembled into virion, transit to the cell surface and mediate cell-cell fusion between infected cells and adjacent, uninfected cells, leading to the formation of syncytial, giant, multinucleated cells. This may contribute to immune escape and favor viral spreading. Ace2R downregulation and subsequent reduction of angiotensin II inactivation and conversion to angiotensin 1-7 trigger a cascade of phenomena, leading to inflammation, humoral and cellular immune reactions, cytokine storm, hyper-viscosity and coagulability, endothelial and parenchymal damage. Wild inferior vertebrates are viral reservoirs, superior vertebrates may be amplifying hosts. Human transmission may occur through respiratory droplets, aerosol, fecal-oral route, direct contact, blood, mother-to-child included [14][15][16][17]. The latency period is generally from 3 to 7 days, with a maximum of 14 days [18], although up to 32 days are reported [19]. Many questions have still not found answers. Why coronavirus was so virulent also in developed countries? Are asymptomatic carriers a source of contagion? How long does immunocompetence last? Will viral mutagenesis rate allow prompt development of effective vaccines?
We report our experience in the CoV2 unit in an Italian, southern community hospital.

Results
At admission, Brescia Cov2 Scale was 0,71 sd 1,34. The most common clinical manifestation was fever (52/89, 58,4%), followed by dyspnea (40/   Hypoxic encephalopathy was present in 36/89 (40%) patients. CoV2 infection was observed in the context of a typical haemorrhagic acute stroke in one patient suffering from arterial hypertension. Two patients had ischaemic stroke during the course of CoV2 SARS. The etiopathogenesis was lacunar in the young, male patient, affected with arterial hypertension, and embolic in the elderly, female patient, suffering from permanent atrial fibrillation.
First oro-pharyngeal and/or nasal swab was positive for Cov2 RNA in 64/89 (72%) patients. A second swab was necessary in 8/89 (9%) patients to confirm the diagnosis. In 5/89 (6%) patients the diagnosis was confirmed by the presence of IgM anti-CoV2 at rapid test. In 10/89 (11%) both swab and rapid test were negative, although clinical and radiological findings were highly suggestive of ongoing SARS Cov2. Two patients died before undergoing diagnostic procedures.

Discussion
Although the incidence of CoV2 SARS was high, the mortality was inferior compared to the north of Italy. This was explained by early diagnosis and therapy in admitted patients and screening on all the inhabitants. Low level of environmental pollution contributed to reduced morbidity and better outcomes. Comorbidities in elderly patients negatively interfered with recovery and weighted on the pathological process leading to the exitus. Blood cell counts, together with specific immunological test, may easily discriminate between immune-depressed, highly-contagious and immune-competent, lowcontagious subjects. Nosocomial isolation is crucial for the former. The latter may be early discharged and home-treated, if pauci-or asymptomatic.
The negativity of rapid test may include false negative. Moreover, as acutely observed [22], oro-pharyngeal swab detects the presence of viral RNA, not alive virus. Its negativity does not exclude ongoing infection. The diagnosis must be based on clinical and radiological findings before invasive procedures, as broncho-aspiration and broncho-lavage. These may confirm low respiratory infection, but they are risky, because of potential iatrogenic viral diffusion, and dangerous because of possible mucosal injury. An alternative may be represented by plasma biomarkers of epithelial (surfactant protein-D, soluble Receptor for Advanced Glycation End Products) and endothelial injury (Angiopoietin 2, Intracellular Adhesion Molecule 1). A metanalysis showed that high plasma levels of Angiopoietin 2 and Receptor for Advanced Glycation End Products were associated with an increased risk of Acute Respiratory Distress Syndrome development [23]. IOT in elderly patients is a difficult task because of the heterogeneity of individual pathological findings. Weaning from invasive to non-invasive ventilation is even more arduous. Because of basal, reduced thoracic excursion, gas exchange is impaired, CO2 levels are high and O2 supply may be unsuccessful. Ventilator-associated/ventilator-induced lung injury (barotrauma, volutrauma, atelectrauma, biotrauma) seem to be more related to acute necrosis and underlying apoptosis rather than to applied techniques. Indeed, alveolar epithelial and endothelial damage, immune-dysregulation, intravascular coagulation, neuro-muscular and cardiological impairment may cause brief and long-term pulmonary dysfunction, leading to fibrosis in case of survival, irreversible pulmonary failure, death.
Plasma levels of CRP, PCT and cytokines, as Interleukin 6, may be useful for monitoring the course of the disease. However, the inflammatory cascade may be followed by lymphocyte exhaustion and immune-depression. Microbiota and innate humoral immune response, firstly represented by mucosal IgA production by B lymphocyte, are the first line of host defense. They play a pivotal role in limiting viral anchorage and invasion. The activation of viral sensors, as system of pattern recognition receptors generates a positive feedback loop, recruiting immune cells, stimulating cytokine production [24]. Specific humoral and cellular immunity, cytokine storm and natural killer cytotoxicity may quickly eliminate the virus. If humoral immune response fails and adaptive immune response against CoV2 does not mount and/or is overwhelmed by viral persistence and CoV2 succeed in hijacking the host cellular machineries, tissular damage occurs. Autoimmune reactions may be triggered and may worsen the clinical picture. Apoptosis may be observed, as host response to limit viral replication. This may involve not only respiratory tract, but also mucosal, intestinal, kidney tubular, neuronal, immune cells, leading to loss of immune-competence, viral spreading and diffuse organ dysfunction [25]. Genetic factors may predispose to the disease. Lymphopenic state or lymphocyte exhaustion may account for infection with other agents or reinfection with CoV2, loss of tolerance, activation of autoimmune cells because of molecular mimicry, epitope spreading and bystander activation [22]. SARS-CoV2 epitopes shares homologies with human heat shock proteins [26] and brainstem respiratory pacemaker [27]. Moreover, strong immune cross-reaction was found between spike protein antibodies and parenchymal proteins, as pulmonary surfactant, mitochondrial proteins and myelin basic protein [28][29][30]. The duration, the magnitude, the type and the term memory of immune protection are unknown. It is also conceivable that host-virus interaction may result in a latent "cold-war", which may attenuate or reinforce virulence, according to genetic factors and immunological asset. The disease may evolve toward a chronic, progressive, multi-organ parenchymal damage and pulmonary thrombo-embolisms. The presence of non-neutralizing or sub-neutralizing concentrations of anti-Cov2-antibodies, may cause an antibody-dependent enhancement (ADE) of viral infection and replication by antibody interaction with cell surface Fc receptors, virus-antibody immune complexes internalization, virion uncoating and release of viral genome in the cytoplasm. The antiviral immune response itself may be downregulated by altered host intracellular signaling pathway. Memory to cross-reactive antigens may have been elicited by previous other coronavirus infection ("original antigenic sin") and account for both less effective, specific immune response to CoV2 and ADE. On the other hand, naïve, specific immune response may efficiently defend children from severe disease. It is still not known whether the anticipatory immunity plays a pivotal role against CoV2 and trained immunity is protective [31].
Radiological findings suggest that consolidations represent frank, inflammatory foci. The peculiar ground-glass areas may be the expression of early microvascular involvement, followed by necrotic phenomena, with possible faster, further evolution toward honey-comb and reticular aspects of fibrosis. In order to prompt decision making on treatments, the key for correct imagings' interpretation is firstly defining the prevalent emphysematous-like or congestive-like pattern, especially in elderly patients. The presence of halo sign may represent peripheral oedema in still savable tissue (penumbra), surrounding the parenchymal lesion. Reversed halo sign may be predictive of worst prognosis because of an ischaemic core with ongoing, peripheral, inflammatory progression. Once resolved, bubble sign may remain. In some patients, the ground-glass lesions may be more evident peripherally (subpleural location) in early phase, because of vasculitis in microvessels, followed by proximal parenchymal extension in fullblown disease. In elderly, lymphopenic patients, the inflammatory picture may paradoxically and apparently be less severe, because of early alveolar loss and rarified pulmonary structure.
CoV2 has multiple targets. Beside non-specific complications of systemic disease, viral spreading may cause peculiar, organ-specific, clinical pictures. Central Nervous System may be involved by direct haematogenous diffusion or through retrograde neuronal route, infectious and para-infectious pathological phenomena, generating a cascade of deleterious, organ-specific and systemic impairment with brief and long-term sequelae [32]. Similar to immune cells, sensory neurons express receptors for pathogen-associated molecular pattern. Inflammatory responses may perturb neuronal activities and elicit pathological reflexes [33]. Both acute and chronic respiratory failure may accentuate neuropsychiatric features, cause acute strokes and accelerate the onset of neurodegenerative diseases [34]. Younger age, embolic genesis, worse prognosis were described in stroke patients [35]. Anti-phospholipid antibodies were detected [36]. Guillan-Barrè like syndrome in CoV2 patients may rapidly evolve to respiratory failure. Cases of lacunar strokes, acute necrotizing encephalitis, seizures, meningo-encephalo-myelitis, myasthenic signs and rhabdomyolisis are reported. The presence of neurological signs worsens the clinical picture of SARS-CoV2, as shown by altered haematological parameters [37]. Hypercoagulable/hyperviscosity state is present. Inflammation in microvessels may result in lacunar ischaemic sufferance and large infarction, because of plaque vulnerability and myocardial damage [38]. The same pathological mechanisms may account for cardiological dysfunctions, mainly represented by acute coronary syndrome and arrhythmias. The latter may be recorded in case of myocarditis, caused by direct viral injury, and be iatrogenic, induced by chloroquine or hydroxychloroquine. Dilated cardiomyopathy and heart failure may ensue [39]. Renal involvement, ranging from mild-moderate proteinuria to acute renal failure, further worsen clinical conditions and may be linked to poor prognosis, long-term complicances and death [40]. These are related to direct viral infection, inflammation, hypercoagulation, volume depletion, heart and respiratory failure, rhabdomyolysis. Beside tubular, we suspect early, acute, glomerular damage.
Current knowledge on CoV2 multiorgan damage is still partial. Reduced number of patients come to observation in emergency unit for fear of CoV2 contagion. Acute neurological as well as cardiological patients are directly transferred to non-CoV2 units. CoV2 related dysfunctions may be misdiagnosed because of previous positive case history of organ impairment and non-urgent examinations are postponed for reducing the risk of in-hospital cross-infection. On the other hand, the delay in diagnosis of all the other diseases endangers public health.
Immune-depressed patients, affected with severe diseases may be more vulnerable also to low viral load, become intermediate reservoirs of more virulent CoV, as suggested by nosocomial outbreaks and poor prognosis. Indeed, lymphopenia is observed at admission and reported in full blown disease [41,42] as well as an increased risk of coinfections [43]. Moreover, both morbidity and lethality increase by aging.
In immunocompetent subjects, human to human transmission may be linked to high viral load, stressors, as hard work shifts, smoking, alcohol or other substance abuse, unhealthy behaviors [44], subsequent increased disease susceptibility, plausibly followed by immune-defiance, as suggested by disease clusters in seafood, wet market workers in China, slaughterhouse workers in Deutschland, express couriers in Italy. The stress gateway reflex induces cerebral micro-inflammation, excites neural pathways, disturbing functions of organs in the periphery [33]. Viral load, mutagenesis rate, crossing of species barrier are expression of contagion and diffusion in high density population, in case of extreme social and economic gap, urbanization, poor hygiene conditions, as in rural farming [45], paucity of environmental resources, conflicts, migrations, climate changes and environmental disasters. These may represent a Damocles' sword for future generation, if shared rules are not respected.
The ability to recombine using both homologous and nonhomologous recombination is known to favor viral evolution and virulence [46,47]. CoVs are recognized among the most rapidly evolving viruses, owing to their high genomic nucleotide substitution rates and recombination, antigenic drift and shift [48]. This may hinder the production of an effective vaccine or render it useless. On the other hand, higher the viral homology, more likely the immune cross-reactivity develops. Neutralizing antibodies may be produced in experimental animals and administered during acute phase, but the risk of thrombotic events may be further increased as well as the antibody-dependent enhancement of viral infection. The latter risk may be reduced by substitution or removal of viral enhancing epitopes. Alternative biotechnological approaches may be blockage of viral anchorage by soluble receptor binding domains of S protein, antibody or single chain antibody fragment against ACE2, target the coronavirus virions directly by using the ACE2 extracellular domain binding the spike protein (ACE2-Fc construct). Lastly, small interfering RNA (siRNA) or antisense oligonucleotides (ASO) to combat the virus by targeting its RNA genome may be generated [49]. However, the availability of these therapies may be limited because of the high cost of production. Administration of antiviral agents may be limited. An affordable therapy may be plasma infusion. It is unknown whether asymptomatic subjects are "super-spreaders", although their immunologic system actively fight and defeat viral attack, reducing virulence more efficiently than any other available drug. They may represent the ideal plasma donors, because of polyclonal antibody immune response to different viral antigens, devoid of autoimmune bioproducts. The study of herd immunity may highlight the mechanisms of immune-resistance. The latter may be overcome by environmental contamination. Beside respiratory droplets, self-inoculation, if hygienic procedures are not respected, and shedding by infectious materials (sputum, fecis, blood) may occur [50]. It is reported that CoV2 resists on surface (aerosol 3 hours, copper 4 hours, cardboard 24 hours, plastic 2-3 days, stainless 2-3 days) [51]. Although, CoVs seem to be able to persist in the environment up to 9 days, they can be inactivated within one minute by disinfection with ethanol, hydrogen peroxide, sodium hypochlorite [52]. Then, the viral load is presumably proportional to immune-depression in amplifying hosts.

Conclusions
Advanced age, comorbidities, lymphocytopenia and bacterial superimposed infections are red flags predicting worse prognosis in SARS CoV2. Healthy behavioral habits safeguard young and middle age subjects from the disease. The most powerful strategy for disease eradication is health education. It is mandatory not to disappoint trust on health care. Elementary rules, as use of caps, googles, gloves, washing hands, disinfecting surface, avoiding crowds and travel, social distancing are important for limiting the risk of infection. Home confinement and lockdown are extreme measures which must be rationally applied, because of long-term deriving economic crisis, due to reduction of gross national product, low tax incomes, need to ensure social support to indigent people, insurmountable account deficit. Protective devises for health care providers must be smartly used, because they are not ecologically disposable. Green (no-contaminated) and red (contaminated) paths must be clearly marked and only health care personnel, charged of daily clinical evaluation of patients, must be adequately dressed. Because of pleiomorphic clinical and radiological findings, it would be better to avail tests for the simultaneous detection of common viral and bacterial agents for the most appropriate therapy, as multiplex PCR array. CoV2 Prognostic Scale may speed up decision making on clinical setting and treatments. International cooperation is fundamental for a rational, concerted action directed to safeguard public health and reduce the impact on socio-economical asset.

Conflict of interests
None.