Coronavirus disease 2019, the new public health emergency that originated in China, is spreading rapidly across the globe with limited tools to confine this growing pandemic. The virus, severe acute respiratory syndrome coronavirus 2, is transmitted by droplet infection from person to person. Our current understanding of the disease spectrum is limited. The proportion of infected children is significantly less compared to adults with the majority of them showing mild symptoms. More than half of symptomatic children present with fever and cough. However, the extent of asymptomatic infection in children and the role they play in community transmission is still undetermined. Although there are case reports of neonates infected with severe acute respiratory syndrome coronavirus 2, vertical transmission from infected mother to new-born is yet to be proven. The disease is confirmed by demonstration of the virus by real-time reverse transcriptase-polymerase chain reaction in respiratory secretions. Due to the lack of specific antiviral agents, we rely on infection-control measures to prevent disease spread and on supportive care for infected ones. This article has summarized the clinical characteristics of children with coronavirus disease 2019 based on published case reports. After it's outbreak in Wuhan City of China in December 2019, it has rapidly spread to the rest of the world and the World Health Organization (WHO) has declared it as a pandemic on March 11, 2020. Our understanding of COVID-19 is evolving along with increasing numbers of cases. SARS-CoV-2 is a newly emerging contagious pathogen without pre-existing immunity in humans. Available data show that the effects on children are less severe than those on adults. Most of the pediatric cases are family cluster cases having epidemiological links to adults.23 Coronaviruses (CoVs) are enveloped, positive-stranded Ribonucleic Acid (RNA) viruses with crownlike appearance under an electron microscope due to the presence of spike glycoprotein.4 CoVs can be divided into four genera: alpha, beta, delta, and gamma, of which alpha and beta CoVs are known to infect humans.5 Till date, seven human coronaviruses (HCoVs) have been identified i.e., HCoV 229E, NL63, OC43, and HKU1, SARS-CoV, MERS-CoV, and SARS-CoV-2. The first four coronaviruses are endemic globally and account for 10% to 30% of self-limiting upper respiratory tract infections in immunocompetent individuals.3 SARS-CoV-2 is sensitive to ultraviolet rays and heat, and can be effectively inactivated by lipid solvents including ether (75%), ethanol (60-90%), chlorine-containing disinfectant, peroxyacetic acid, and chloroform except for chlorhexidine.3 The virus has been shown to use the angiotensin-converting enzyme 2 (ACE2) for cell entry similar to the severe acute respiratory syndrome (SARS) coronavirus.6SARS-CoV-2 belongs to the ß-coronavirus genus.9 The intermediate host may have a role in causing recombination and mutation in the virus with the expansion of genetic diversity. Researchers in China identified the pangolin as one of the potential sources of SARS-CoV-2 because genetic sequences of viruses isolated from the scaly animals were 99% similar to that of the circulating virus.8 Later, Xia hypothesized that SARS-CoV-2 could have evolved in canid mammalian intestine or tissues associated with the intestine.10 However, the actual origin of the virus remains unclear.Genomic analyses suggested that the SARS-CoV-2 probably originated from bats as the virus had 89% to 96% genomic identity with bat SARS-like coronavirus and was transmitted to humans by some other intermediary host.12 Infection is transmitted by inhalation of large droplets generated during coughing, talking, or sneezing by both symptomatic and asymptomatic persons within close range (within six feet). Infection can also be acquired by touching surfaces contaminated by these droplets and then touching the nose, mouth, and eyes.4 Transmission through aerosols can also occur under specific circumstances such as tracheal intubation and extubation, noninvasive ventilation, manual ventilation before intubation, cardiopulmonary resuscitation, bronchoscopy, administration of high-flow oxygen or nebulized medications, tracheotomy and upper gastrointestinal endoscopy.13 The infected droplets can spread one to two meters and deposit on surfaces where it remains viable for a variable period. The virus was detectable up to four hours on copper, up to 24 hours on cardboard, and up to two to three days on plastic and stainless steel surfaces.14 The airborne spread has not been reported so far.At first, animal to human transmission was assumed to be the main mechanism of disease spread as the initial cases were linked to the direct exposure to the Huanan seafood wholesale market of Wuhan. Subsequent cases were not associated with this exposure. Person to person transmission of the disease has been confirmed by the studies.15 The viral load detected in the nasal swab of the asymptomatic patient was similar to that in the symptomatic patients.16 However, the viral load in nasopharyngeal swabs can vary according to the disease severity, severe cases having significantly higher (~60 times) load than that of mild cases.17 The study done by Xu Y, et al. failed to demonstrate the replication-competent virus in fecal swabs, which is required to confirm the potential for fecal-oral transmission.18 On contrary to this, live SARS-CoV-2 has been detected in a stool sample of a few patients.15 According to a joint WHO-China report, the fecal-oral transmission did not appear to be a significant factor in the spread of infection.9 Hence, the fecal-oral route of transmission is yet to be established. Viral shedding in the respiratory specimen is also found to be longer in children with COVID-19.12 Prolonged fecal and nasal secretion of the virus in children has substantial implications for community spread.The virus has been detected in specimens like bronchoalveolar lavage fluid, sputum, nasal swabs, pharyngeal swabs, feces, and blood with decreasing frequency.19 All the neonatal specimen tested including some placentas were negative for the virus. However, Zeng, et al. reported early onset SARS-CoV-2 infection in three out of 33 neonates born to mothers with COVID-19.20 All three neonates tested positive for the virus in nasopharyngeal and anal swabs and had chest radiologic image of pneumonia. The vertical maternal to the fetal transmission of the disease could not be ruled out in a given cohort as the source of SARS-CoV-2 in the neonates could be of maternal origin. Likewise, there are case reports of three neonates with elevated SARS-CoV-2 immunoglobulin M (IgM) in the blood collected within few hours of birth who were born to mother with COVID-19.22 The elevated IgM antibodies suggested that the neonates were infected in utero. However, nasopharyngeal swabs of all three neonates were negative by RT-PCR. Additional examination of maternal and newborn samples should be done to confirm this preliminary observation.Controversy exists regarding whether SARS-CoV-2 can be transmitted in utero from an infected mother to her infant before birth. In one study analyzing 38 pregnant women with COVID-19 and their newborn in China, there were no confirmed cases of intrauterine transmission of SARS-CoV-2 from mothers to their fetuses.23 Newborns may still acquire a SARS-CoV-2 infection through close contact with infected mothers. Thus, mothers with confirmed or suspected COVID-19 should take all precautions to prevent transmission to the infant during feeding. Considering the benefits of breastfeeding and the insignificant role of breastmilk in the transmission of other respiratory viruses, United Nations Children's Fund (UNICEF) has recommended continuing with breastfeeding while applying necessary precautions to prevent transmission of infection.It is unknown whether SARS-CoV-2 can be transmitted through breast milk. The report of testing breast milk samples of six mothers with COVID-19 found no virus in the maternal milk.2 People are thought to be most contagious when they are most symptomatic. Symptoms of COVID-19 are varied and the disease presentation can range from no symptoms (asymptomatic) to severe pneumonia with multi-organ dysfunction and death.9 A significant proportion of children with COVID-19 do not appear to develop any symptom, or have subclinical symptoms. Relatively few children are hospitalized, and fewer children than adults experience fever, cough or shortness of breath, according to the report from the Centers for disease control and prevention (CDC). Among pediatric patients, children under age one year, and children with underlying health conditions are at the highest risk of serious illness resulting in hospitalization.24 Chronic lung disease (including asthma), cardiovascular disease, and immunosuppression are common underlying health conditions in children with COVID-19. Clinical features in symptomatic children tend to be mild and they recover within one to two weeks after the onset of illness.25The incubation period of 2019-nCoV infections ranges from one to fourteen days.8 Dong, et al.8 reported the proportion of severe and critical cases for the age groups <1, 1-5, 6-10, 11-15, and >15 years as 10.6%, 7.3%, 4.2%, 4.1%, and 3.0% respectively. The most common presenting symptoms include fever followed by cough in more than half of symptomatic children. Upper respiratory tract symptoms such as rhinorrhea and sore throat are also relatively common and it is not uncommon for children to have diarrhea and/or vomiting, even in some cases as their sole presenting features  including 22,512, 72,314 and 149,082 cases respectively showed the incidence of 1.2%, 2%, and 1.7% among patients aged <18 years respectively.40Trained immunity refers to the use of certain vaccines such as Bacille Calmette-Guerin (BCG) to train innate immunity to generate immune memory. It is speculated that children were less sensitive to the virus because the maturity and function of ACE2, receptor for SARS-CoV-2 in children may be lower than that in adults.8 The proportion of severe and critical cases was highest in the age group of less than one year. There are only a few reported deaths among children due to COVID-19.The largest review of children with COVID-19 to date including 2143 children in China reported severe cases (defined as hypoxia) in 5.6% children and critical cases like acute respiratory distress syndrome (ARDS)/multiorgan dysfunction syndrome in 0.6% children.The white blood cell counts can vary in children with COVID-19. Lymphopenia and leukocytosis are relatively uncommon in children, with raised lymphocyte counts and diminished total white cell count present in 17-22% of children .43 Inflammatory markers such as C-reactive protein (CRP) and procalcitonin, liver transaminases, and D-dimers are often raised only very mildly in children and are not common in children as compared with the adults.These findings are different from adults where lymphopenia appears most common.Radiologic features in children also differ from their adult counterparts. Based on published case reports on children with COVID-19, radiological evidence of pneumonia is present in up to two-thirds of children irrespective of symptoms . Changes27 Lung consolidation may occur in severe cases.2Lesions were bilateral in up to two-thirds of cases with abnormal CT scans. Fifty percent of chest CT scans had consolidation with a surrounding halo in one case series.45 These definitions are based on the currently available information and are regularly revised as new information accumulates.Any suspected case should be tested for the infection with SARS-CoV-2 using a molecular test. A Suspect case is: i) a patient with acute respiratory illness, and a history of travel to or residence in a location reporting community transmission of COVID-19 disease during the 14 days before symptom onset; or ii) a patient with an acute respiratory illness and having been in contact with a confirmed or probable COVID-19 case in the last 14 days before symptom onset, or iii) a patient with severe acute respiratory illness, and requiring hospitalization, and in the absence of an alternative diagnosis that fully explains the clinical presentation.46 Alternative specimens include oropharyngeal swabs, sputum (if produced), and endotracheal aspirate or bronchoalveolar lavage from patients who are intubated. SARS-CoV-2 RNA is detected by reverse-transcription polymerase chain reaction (RT-PCR), a positive test of which confirms the diagnosis. If a negative result is obtained from a patient with a high index of suspicion for COVID-19, particularly when only upper respiratory tract specimens were collected, additional specimens, including from the lower respiratory tract if possible, should be collected and tested.47The CDC recommends collection of a nasopharyngeal swabs to test for SARS-CoV-2.32 Several investigational agents are being explored for the treatment of COVID-19. These include redemsivir , interleukin-6 pathway inhibitors , convalescent plasma, favipiravir (an RNA polymerase inhibitor), and chloroquine/hydroxychloroquine  among many others. More evidence is needed before these drugs are recommended.Treatment is essentially supportive and symptomatic depending upon the severity of the disease. Neither antiviral agent nor immunomodulatory therapy with exact efficacy is available till now to be used in children with COVID-19. Antiviral therapy , interferon-D, corticosteroids, and intravenous immunoglobulin have been used to treat children.48 Mechanical ventilation may be needed in patients with ARDS. Evidence-based treatment guidelines for ARDS should be followed that includes conservative fluid strategies, empirical early antibiotics for suspected bacterial co-infection, lung-protective ventilation, prone ventilation, and consideration of extracorporeal membrane oxygenation for refractory hypoxemia. Septic shock and specific organ dysfunction appear to occur in a significant proportion of patients with COVID-1 9-related critical illness and are associated with increasing mortality.48In hypoxic patients, the provision of oxygen through nasal prongs, face mask, high flow nasal cannula, or non-invasive ventilation is indicated. Antibiotics and antifungals are required if co-infections are suspected or proven. Management of severe cases is similar to the management of most viral pneumonia-causing respiratory failure.49 Type L patients can be ventilated with higher volumes  without the risk of ventilator-induced lung injury due to high lung compliance whereas type H patients are treated the same as ARDS.COVID-19 pneumonia is thought to be a specific condition characterized by severe hypoxemia and nearly normal lung compliance with two phenotypes: Type L  and Type H .50 It may be related to a positive non-specific immune response like increased secretion of pro-inflammatory cytokines like IL-1B triggered by the BCG vaccination that has antiviral immunity.An epidemiological study has shown that countries without universal BCG vaccination have been severely affected both in terms of mortality and spread of infection compared to countries with universal and longstanding BCG policies.In the absence of specific therapy directed against the virus, it is of paramount importance to implement infection control practices by controlling the source of infection, blocking the route of transmission, and protecting the susceptible population. Prevention involves isolation of suspected and confirmed cases, and strict infection control measures at hospitals that include contact and droplet precautions. Non-specific manifestations of the disease, infection transmission both before the onset of symptoms and after clinical recovery, long incubation period, tropism for mucosal surface, and prolonged duration of the illness makes the infection prevention and control measures very challenging. Appropriate social distancing and daily preventive behaviors are of utmost importance for all age groups.COVID-19 is a novel disease with an incompletely described clinical course, especially in children. Children are mainly infected with SARS-CoV-2 from their family members but less severely than adults, presenting mild symptoms, and a good prognosis. It is necessary to learn as much as possible through observational studies and clinical trials across a wide range of patients, populations, and health care settings.None.


Human beings are currently experiencing a serious public health event. Novel coronavirus disease 2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), has infected about 3 million people worldwide and killed more than 200,000, most being the elderly or people with potential chronic diseases or in immunosuppressive states. According to big data analysis, there are many proteins homologous to or interacting with the angiotensin-converting enzyme 2 (ACE2), which, therefore, may not be the only receptor for the novel coronavirus; other receptors may also exist in host cells of different species. These potential receptors may also play an important role in the infection process of the novel coronavirus. The current study aimed to discover such key proteins or receptors and analyze the susceptibility of different animals to the novel coronavirus, in order to reveal the transmission process of the virus in cross-species infection. We analyzed the proteins coded by the ACE2 gene in different mammalian species and predicted their correlation and homology with the human ACE2 receptor. The major finding of our predictive analysis suggested ACE2 gene-encoded proteins to be highly homologous across mammals. Based on their high homology, their possibility of binding the spike-protein of SARS-CoV-2 is quite high and species such as Felis catus, Bos taurus, Rattus norvegicus etc. may be potential susceptible hosts; special monitoring is particularly required for livestock that are in close contact with humans. Our results might provide ideas for the prevention and control of the novel coronavirus pneumonia. Emerging infectious diseases (EIDs) pose a risk to global public health and biosafety. Over 5,000 viruses have been identified to date, of which ~75% are of a zoonotic origin, and can cross the species barrier and establish infection in human beings . Since D-coronavirus genus of the family Coronaviridae. The coronaviruses infecting human beings at present had originated from animals, and their natural hosts are generally Chiroptera (bats) and rodents (rats)  . Additios (rats) . WhetherAngiotensin-converting enzyme (ACE) is a monomeric, membrane-bound, zinc- and chlorine-dependent dipeptidase . It can The receptor binding domain of SARS-CoV-2 shares high sequence homology with SARS-CoV, indicating the potential binding of ACE2 with SARS-CoV-2 . The difIn this study, protein sequences corresponding to the ACE2 gene were downloaded from UniProt database , with suNyctereutes procyonoides (Raccoon dog), Neophocaena asiaeorientalis (Finless porpoise), and Rhinolophus sinicus (Chinese rufous horseshoe bat) showed the complexes to have highly similar overall structures . If there were multiple identical protein sequences encoded by the ACE2 gene in each species, a sequence was randomly selected as the representative sequence of the species for subsequent processing. The screened sequences were aligned using Clustal Omega on the EBI web server . MaximumAfter screening, sequence similarity and identity were analyzed again to study further the relationship between the protein sequences encoded by the ACE2 gene. The key operation process can be divided into the following steps: The protein sequences from the source host that were not mammals were manually deleted, while the remaining protein sequences were compared with the ACE2 protein sequence encoded by the human ACE2 gene one by one using the Needleman-Wunsch algorithm , and theNyctereutes procyonoides, Neophocaena asiaeorientalis asiaeorientalis, and Rhinolophus sinicus was modeled with Coot (The SARS-CoV-2 S-RBD in complex with ACE2 from ith Coot  using thith Coot  as the tith Coot  with an https://www.uniprot.org/.Publicly available datasets were analyzed in this study. This data can be found here: YC and JL conceived of the research, prepared the manuscript, and completed its revision. YC and YS performed data analysis. XT and ZB polished and finalized the figures. YG, WL, JQ, and DL provided various suggestions. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The ongoing Corona virus (COVID-19) pandemic has witnessed global political responses of unimaginable proportions. Many nations have implemented lockdowns that involve mandating citizens not to leave their residences for non-essential work. The Indian government has taken appropriate and commendable steps to curtail the community spread of COVID-19. While this may be extremely beneficial, this perspective discusses the other reasons why COVID-19 may have a lesser impact on India. We analyze the current pattern of SARS-CoV-2 transmission, testing, and mortality in India with an emphasis on the importance of mortality as a marker of the clinical relevance of COVID-19 disease. We also analyze the environmental and biological factors which may lessen the impact of COVID-19 in India. The importance of cross-immunity, innate immune responses, ACE polymorphism, and viral genetic mutations are discussed. The symptoms of COVID-19 range from mild to severe, and include mainly fever, cough, and respiratory distress. Severe cases with pneumonia and hypoxemia result in considerable mortality. The highest rate of infection has been seen in adults and aging individuals, but newborns and children could also be infected by SARS-CoV-2. Older individuals with pre-existing chronic health condition shave a higher risk of developing severe complications after SARS-CoV-2 infection. COVID-19 has emerged as a pandemic of respiratory illness ever since the first cases appeared in Wuhan, China, in December 2019 [th March 2020. Only essential services would be functioning and government services apart from health, law and order, banking, power and a few others have been suspended altogether. While the response by the Indian government both at the central and state levels has been commendable and timely given the global COVID-19 crisis, we deliberate some of the other reasons why the authors believe COVID-19 may have a lesser impact on India.This ongoing pandemic has witnessed global political responses of unimaginable proportions. Many nations have implemented lockdowns that involve mandating citizens not to leave their residences for non-essential work. The rules of lockdown vary from country to country. The Indian Prime Minister announced a nation-wide lockdown from the midnight of 24th rank among nations for the number of diagnosed COVID-19 cases (www.worldometers.info/coronavirus/). The first case of COVID-19 was detected in India in the southern state of Kerala on 30th January 2020 and was a medical student who had returned from Wuhan. Two other cases, also medical students back from Wuhan soon surfaced from Kerala . The Indian government implemented thermal screening of inbound international air-travelers. Such measures were implemented for China around17thJanuary and then, in a stepwise fashion, expanded to include all international passengers (https://pib.gov.in/PressReleaseIframePage.aspx?PRID=1599901). Interestingly, thermal screening is far from a fool-proof way to detect SARS-CoV-2 carriers, many of whom may be in the middle of the incubation period or mild cases and hence asymptomatic. Further, the quarantine of international travelers on arrival was initiated only for those from China and its immediate neighbors at the beginning and then expanded for travelers from other countries, that too in a phased manner, while observing global trends of COVID-19 spread. Domestic travel in India continued unabated without incorporating any screening. Further, the initial testing criteria with confirmatory qRT-PCR (quantitative reverse transcription polymerase chain reaction)-based tests for suspected patients, released by the Indian Council of Medical Research (ICMR), emphasized testing only those with laboratory-proven contacts or recent foreign travel (www.icmr.nic.in/content/covid-19). With such restrictions, the movement of several SARS-CoV-2 positive patients, whether asymptomatic or symptomatic, must have occurred all over India. However, 9 weeks after the first case was diagnosed, India has a COVID-19 count of 2300 (www.mohfw.gov.in/). In contrast, the first two Italian cases were Chinese tourists who arrived in Milan on 23rd January, and tested positive on 30th January, the same time when the first Indian case was recorded [st when daily more than 250 new cases (more than 500 new cases daily since 4th March) were diagnosed (https://ourworldindata.org/coronavirus). It is curious that among the two countries which reported their first case at almost the same time, the progression has been so disparate. One may argue that testing in India has been sluggish in contrast to aggressive testing by some developed nations. However, India had tested samples  from 26798 patients suspected to have COVID-19, as of 27th March which still gave a testing positivity rate of 2.6%, a figure which has not increased significantly over time or with changes in testing criteria (https://icmr.nic.in/sites/default/files/whats_new/ICMR_website_update_27March_9AM_IST.pdf). A further update by the ICMR pegged the figure at 42788 samples by 30th March (2.5% test positivity). In comparison, Italy at the top of the spectrum had carried out nearly 206886 tests as of 20th March with 41035 positives, which translates into a positivity rate of tested individuals of 19.8%. South Korea, on the other hand, adopted a comprehensive strategy of community testing. Such an approach translated into 8652 positives in 316664 tests (2.7%) (www.statista.com/statistics/1028731/covid19-tests-select-countries-worldwide/). A few other countries at different ends of the spectrum are represented in the graph in The world’s second-most populous country lies around the 402, which is higher than the six countries with maximum COVID-19 cases. Such a finding is a paradox as countries with higher densities and people staying closer to each other are theoretically at a higher risk of contracting communicable diseases transmitted by fomites or aerosols. Similarly, the examples of South Korea (517 persons/km2) and Japan (333 persons/km2), which have a relatively lower incidence of COVID-19 and a higher population density, support this curious case. Trying to explain this paradox would require further research.The population density of India is 414 persons/kmIn the Köppen classification of climatic zones of the world, both Wuhan, the apparent epicenter of the pandemic and most of Italy are categorized as type C (mild temperate) whereas much of India has either a dry (type B) or tropical climate (type A). However, this again raises queries on why Iran having a dry desert climate (type B) bore the brunt of COVID-19 cases . It has Like other animal and human corona viruses, SARS-CoV-2 is also a positive-strand RNA (approximately 30kilobases) virus which codes for a replicase enzyme associated with other enzymatic activities and several structural proteins of which the spike protein (S) is essential for viral entry into host cells . SARS-CoThe defense against any viral infection involves both innate immunity and adaptive immunity. While adaptive immunity relies on antibodies and T-cells which can recognize viral antigens with high degrees of specificity, the innate immunity utilizes receptors  which recognize broad structural motifs present in bacteria or viruses but generally absent in host cells. There are currently no established data on the specific role of either humoral or cellular immunity or innate immunity in patients recovering from COVID-19, but immune response associated with SARS-CoV and MERS-CoV has been studied in some detail ,11. AlthIt is noteworthy that many corona viruses are widely present in cattle, pigs and chickens producing a variety of diseases affecting the respiratory and gastro-intestinal systems in these species . The aniAnother interesting observation is the apparent exclusion of malaria-endemic zones by the COVID-19 epidemic. India, large parts of Africa, and parts of South America that report ongoing malaria transmission have had a low incidence of COVID-19. The reasons are not known, but an explanation may lie in the ensuing cytokine storm, that may be involved in the severe respiratory manifestations of COVID-19 . It is nvis-à-vis the outcome of COVID-19 will require more time [Apart from cross-immunity, an essential mechanism of the variable outcome of COVID-19 may lie in the polymorphism of ACE2 protein, which is coded by a gene on the X-chromosome. Several single nucleotide polymorphisms of this protein have been described which may have implications in altered expression levels of ACE2 or its interaction with S protein of SARS-CoV-2. However, a detailed analysis of such polymorphisms in different populations ore time .While not directly relevant to issues discussed in this article, a possibility also exists that many cases may be missed due to the sensitivity of the tests being performed. Mostly pharyngeal and nasal swabs are used for qRT-PCR based diagnostics. A recent article mentions these samples as having only 32% and 63% positivity while testing for COVID-19 .www.epicentro.iss.it/coronavirus/bollettino/Report-COVID-2019_20_marzo_eng.pdf).Another vital aspect of COVID-19 is mortality in diagnosed cases. Mortality is the statistic that is most relevant for developing nations such as India. Acute viral illnesses as well as acute infections due to any pathogenic agent, are prevalent in India, owing to dismal standards of hygiene, especially among a large section of its population which lives below the poverty line. An exact number cannot be ascribed to the incidence of viral infections, because of the sheer diversity of such infections ranging from respiratory viral infections  to several arthropod-borne viral fever syndromes. The diagnostic facilities are non-existent in the periphery, and the majority of these infections may be subclinical or may present with atypical manifestations . An ideaEven in India, more than half of the deaths that have ensued have affected patients more than 60 years of age, the accepted geriatric age cutoff in the country. Although the chart details of the Indian patients are unavailable, the available data collected from reputed news sources is presented in www.worldatlas.com/articles/countries-with-the-largest-aging-population-in-the-world.html). In life-expectancy it also ranks second after Japan among all countries with a population more than 10 million. Italian healthcare provides for universal coverage and is largely free. What this means is a sizeable section of the population in Italy has survived to an age that would be impossible for them to do up to, in other countries. Contrast this to the situation in India where healthcare services do not have this much peripheral penetration. Further, transport and awareness issues as well as societal preferences for the younger earning population, results in neglect of the elderly and failure of free healthcare to translate into health gains for the older population. India’s life expectancy stands at 69 years, interestingly less than the mean age of COVID-19 fatalities in Italy. So, India may not have the most vulnerable age group for COVID-19 to exert its effect to the fullest. Overall, if one analyzes the mortality statistics of India in comparison to other countries  stands second in the world after Japan (27%) in having a population of > 65 years age (ountries , it is countries .The different restrictive measures adopted by the central and the state governments in India like visa and travel restrictions, home isolation and quarantine, and finally nation-wide lockdown are classical methods of prevention of the community spread of SARS-CoV-2. These methods must have played a role in limiting the effect of this pandemic on the Indian population. However, we strongly feel that various biological and environmental factors have also contributed significantly to this process, which may result in COVID-19 in India behaving more mildly in contrast to its global effects. The careful analysis of such biological factors will certainly open new avenues to combat outbreaks of novel viruses, through research on cross-immunity, immuno-modulation and perhaps life-style management and dietary manipulations.


In the obese, the evidence for the choice of the optimal emergency front-of-neck access technique is very limited and conflicting. We compared cannula and scalpel-bougie emergency front-of-neck access techniques in an anaesthetised porcine model with thick pretracheal tissue. Cannula and scalpel-bougie cricothyroidotomy techniques were performed in 11 and 12 anaesthetised pigs, respectively. Following successful tracheal access, oxygenation was commenced and continued for 5 min using Rapid-O2 device for cannula and circle breathing system for scalpel-bougie study groups. The primary outcome was a successful rescue oxygenation determined by maintenance of arterial oxygen saturation >90% 5 min after the beginning of oxygenation. Secondary outcomes included success rate of airway device placement, time to successful airway device placement, and trauma to the neck and airway. The success rate of rescue oxygenation was 18% after cannula, and 83% after scalpel-bougie technique (P = 0.003). The success rate of airway device placement was 73% with cannula and 92% with scalpel-bougie technique (P = 0.317). Median (inter-quartile-range) times to successful airway device placement were 108 (30–256) and 90 (63–188) seconds (P = 0.762) for cannula and scalpel-bougie emergency front-of-neck access, respectively. Proportion of animals with iatrogenic trauma additional to the procedure itself was 27% for cannula and 75% for scalpel-bougie technique (P = 0.039). Thus, in the porcine model of obesity, the scalpel-bougie technique was more successful in establishing and maintaining rescue oxygenation than cannula-based technique; however, it was associated with a higher risk of severe trauma. The current Difficult Airway Society (DAS) guidelines recommend the scalpel-bougie cricothyroidotomy as a default technique for emergency front-of-neck access (eFONA) procedure. The guidelines, however, acknowledge that the evidence base for the choice of scalpel technique is limited and that the eFONA may indeed be performed using either a scalpel-bougie or cannula technique . A systeThe Fourth National Audit Project (NAP4) acknowledged that serious airway morbidity is more frequent in the obese; and that eFONA techniques fail frequently in the hands of anaesthetists. The NAP4 specifically noted a high failure rate (63%) of cannula technique, mainly attributed to neck positioning, obesity, poor insertion technique, incorrect cannula ventilation method and inadequate training –15. NeveThe study protocol was approved by the ethical committee for animal experimentation under the administrative agency for Food Safety, Veterinary Sector and Plant Protection of the Republic of Slovenia . The animals were reared in accordance with the Council of the European Union directives on minimum standards for the protection of pigs (2008/120/EC), while the overall execution of the study was in full compliance with the National Institutes of Health’s Guide for the Care and the Use of Laboratory Animals and the ARRIVE guidelines.Sus scrofa domesticus) crossbred from the Landrace and Large White, were obtained from a commercial farm following confirmation of their good general health status. Following the 3R principles , this study was performed in animals that were primarily included in a study of neurological and histological outcomes after subarachnoid injection of a liposomal bupivacaine suspension .Shapiro-Wilk test was used to test continuous data for normal distribution, followed by Student’s t test or Mann Whitney U test. Nominal variables were compared using Fisher’s exact test. Statistical analysis was performed with the GraphPad Prism 8 . The difference was considered statistically significant at Twenty-three animals were enrolled in the study; 11 were randomized to cannula and 12 to scalpel-bougie study groups . The meaThe scalpel-bougie technique was found more successful and in providing rescue oxygenation and ventilation compared to cannula technique (P = 0.003) . In termThe cannula was inserted through the cricothyroid membrane in 7 (64%) animals, between cricoid and first tracheal cartilage in one (9%), while in 3 (27%) animals the entry point was not discernible. The obvious kinking of the cannula was encountered in three cases (27%). In the scalpel-bougie group, the tracheal tube insertion through the cricothyroid membrane was documented in 10 (83%) animals, and in one respective animal (8%) through the cricoid and between the cricoid and the first tracheal cartilages.The scalpel-bougie technique was more traumatic, resulting in two life-ending injuries such as carotid artery laceration and major tracheal bleeding  Tables. In an anaesthetised porcine model, a scalpel-bougie technique was more successful in establishing and maintaining rescue oxygenation as compared to the cannula technique while associated with a higher incidence of severe trauma. No significant differences were observed in the rate and the time to successful device placement between the two eFONA techniques. Since the thickness of pre-tracheal tissue in the pig model coincides with the measurements in the morbidly obese patients, the findings of this study can be extrapolated to the obese patient population .TM catheter with a good memory of initial shape [2 device tubing. The kink-resistant and longer cannulas might thus be of benefit in the obese neck. By contrast, a higher success rate of the scalpel-bougie technique in the obese model could be attributed to the use of the two-finger technique for blunt tissue dissection and cricothyroid membrane fixation before performing a transverse incision. In addition, the scalpel-bougie technique allowed a reliable prediction of oxygenation success from the first effective breath while obtaining a definitive eFONA with safe and secure ventilation [In accordance with NAP4 data, our study reconfirms the low success rate of the cannula technique in the obese, primarily attributed to relative shortness of the cannula with respect to pre-tracheal tissue which increased the risk of cannula displacement and kinking despite using the 14 gauge Insyteal shape . The cantilation . Neverthtilation . Our fintilation  suggestitilation .Our study reconfirmed the impression that the scalpel technique is associated with a higher risk of severe vascular injury ,27,28 whConsistent with the findings in the ´wet´ sheep model of morbidly obese neck, we have not observed any differences in insertion times between the cannula and scalpel-bougie technique . HoweverBody habitus in the severely obese predisposes to greater difficulty encountered when identifying landmarks for cricothyroid puncture, particularly in a time-critical setting . The incIn our porcine model, the mean internal tracheal diameter was approximately 15 mm and comparable to that of the adult human trachea . Dead pi2 of 80% before commencing the eFONA procedure, it was very likely that the controlled lab-environment may not have adequately reflected the variables of clinical emergency circumstances. Second, we slightly modified the oxygen jetting technique using RapidO2 to deliver oxygen every 15 seconds irrespective of SpO2 reading to standardize delivered oxygen volume. Third, performing the same procedure multiple times might have had a learning effect; however, each anaesthetist performed both procedures the same number of times in a random order, reducing potential bias. Fourth, due to small sample size, the results should be interpreted and translated with caution. Further studies with larger sample size are therefore warranted. Fifth, in pigs, the sternohyoid muscle overlays the larynx which could affect the handling and bleeding of the pretracheal tissue. Finally, similar to a recently published study we did not paralyze our animal models [Our study has the following limitations: First, although we waited until the animals desaturated to SpOl models . DespiteIn an obese animal model, the scalpel-bougie technique was more successful in providing rescue oxygenation than cannula technique. However, caution is advised with scalpel-bougie technique due to the higher risk of severe trauma. The results suggest that the pre-tracheal tissue thickness needs to be taken into consideration when deciding on the eFONA technique.S1 TableTime to device placement and results of arterial gas analysis after scalpel-bougi emergency front of neck access at different time points .(DOCX)Click here for additional data file.S2 TableCM Cricothyroid membrane(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableCM Cricothyroid membrane, ND Not discernible(DOCX)Click here for additional data file.


The cluster of differentiation 44 (CD44) and the hyaluronan-mediated motility receptor (RHAMM), also known as CD168, are perhaps the most studied receptors for hyaluronic acid (HA); among their various functions, both are known to play a role in the motility of a number of cell types. In peripheral nerve regeneration, the stimulation of glial cell motility has potential to lead to better therapeutic outcomes, thus this study aimed to ascertain the presence of these receptors in Schwann cells  and to confirm their influence on motility. We included also a Schwann-like phenotype (dAD-MSCs) derived from adipose-derived mesenchymal stem cells (uAD-MSCs), as a possible basis for an autologous cell therapy. CD44 was expressed similarly in all cell types. Interestingly, uAD-MSCs were RHAMM(low), whereas both Schwann cells and dASCs turned out to be similarly RHAMM(high), and indeed antibody blockage of RHAMM effectively immobilized (in vitro scratch wound assay) all the RHAMM(high) Schwann(-like) types, but not the RHAMM(low) uAD-MSCs. Blocking CD44, on the other hand, affected considerably more uAD-MSCs than the Schwann(-like) cells, while the combined blockage of the two receptors immobilized all cells. The results therefore indicate that Schwann-like cells have a specifically RHAMM-sensitive motility, where the motility of precursor cells such as uAD-MSCs is CD44- but not RHAMM-sensitive; our data also suggest that CD44 and RHAMM may be using complementary motility-controlling circuits. The traditional approach to repair an injury of the peripheral nervous system (PNS) is the reconnection of the proximal and distal extremities of the severed nerve, with use of autologous nerve grafting reserved for severe injuries to bridge large defects . In the On the other hand, whether Schwann cells are sourced directly or differentiated from precursors, their efficacy is modulated by their microenvironment, and specifically by their extracellular matrices (ECMs) ,26. In pIn the PNS, ECMs often lack classical matrix proteins , but are rich in hyaluronic acid (HA) and in HA-binding proteins  ,28. HA iXenopus laevis)  was then added to the cell culture, and cell motility from the edge to the center of the scratch was monitored for 24 h with an AS MDW live cell imaging system (Leica) using a (63x/1.30 Plan Apo glycerine) objective, the (BGR) filter set (Chroma (61002)), and a (red (DS Red)) Precise LED fluorescent light source. Imaging software Image Pro 6.3 by Media Cybernetics LtD . Point visiting was used to allow multiple positions to be imaged within the same timecourse and cells were maintained at 37 °C and 5% COSamples were fixed and stained for RHAMM and CD44 as previously described, except for the primary antibody and secondary antibody used to stain CD44 , and α-tubulin 1 h and 24 h after the scratch in order to investigate HA receptors expression and tubulin polymerization of the cells on the edge of the scratch. For the tubulin staining, the cells were fixed as previously described after 1 and 24 h incubation after the wound was created, pre-incubated in medium containing HA or not, and pre-treated or not with anti-RHAMM and/or anti-CD44 as previously described. The cells were then washed and blocked with 5% goat serum in PBS for 1 h at RT, washed further, and permeabilized with 0.5% saponin in PBS for 30 min at RT. The cells were then incubated with primary polyclonal rabbit anti-α tubulin, washed three times with PBS, and incubated with goat anti-rabbit Alexa Fluor 350, RT, in the dark. The cells were washed again three times for 5 min with PBS, and mounted with anti-fade reagent. Pictures of the samples were then processed using same live-imaging microscope as previously described.P-values .Statistical significance of the results of flow cytometry analysis was evaluated by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests using GraphPad Prism 6 . Levels of significance were expressed as The currently available information about the role of HA and its receptors in the PNS are rather scarce. CD44 has been proposed as a glial marker e.g., for retina glial cells (Müller cells) , non-myeSince CD44 expression is widely reported both in AD-MSCs ,60,61 anRegarding RHAMM, literature offers hardly any data about its expression in PNS glia; our analysis indicates an important difference between uAD-MSCs and all other cell types. Firstly, in uAD-MSCs, RHAMM was not detected via immunostaining A and WesTherefore, despite some differences among the analytical techniques, RHAMM was shown to be significantly upregulated upon uAD-MSC differentiation to dAD-MSCs; the latter cells not only express typical glial markers see  but alsoIt is worth pointing out that the number and identity of RHAMM isoforms is variable, and several of them have been recorded in glial cells. For example, in culture, it has been shown that astrocytes produce a 72 kDa isoform, while microglia at least three with molecular weights of 50, 65, and 80 kDa (50 kDa being the major one) . In addiThis study does not focus on the specific identification of the RHAMM isoforms, but it must be noted that RHAMM always presented a double band at ≥90 kDa in dAD-MSCs, nSCs, and aSCs. Often two other isoforms could be detected at about 50 and 70 kDa , but in a more variable fashion. Preliminary experiments may indicate a predominant cytoskeletal localization for the 50 kDa form, and on membranes for the other forms see .(a)The supplementation of the cell culture medium with HA Anti-RHAMM almost completely immobilized the three RHAMM(+) types, but not uAD-MSCs, whose motility was unaffected by this blocking antibody ((c)The effect of anti-CD44 was largely different; this blocking antibody abrogated the motility of uAD-MSC, appreciably reduced that of dAD-MSCs and aSCs  and had marginal influence on nSCs, but with largely variable results. Since the overall levels of CD44  did not appear to be much different among the cell types, the scarce sensitivity of nSCs to anti-CD44 may be due to a specific post-translational modification of this receptor. In any case, the key observation is that also CD44 blockage reduced motility, but through mechanisms quite different from RHAMM blockage. It is noteworthy that the RHAMM(+) cell types had some gain in wound closing speed in the presence of HA, also in the presence of anti-CD44, although the effect was marginal in comparison to the non-blocked controls.(d)Anti-CD44 and anti-RHAMM together had an additive (if not synergic) effect; all cell types were immobilized by their combination, independent of the presence of HA.The key role played by HA receptors in cell motility is well known, although literature does not always report unequivocal effects; for example, blocking RHAMM decreases the migration of smooth muscle cells (after injury)  and of e(a)Anti-RHAMM did not appreciably bind to uAD-MSCs, thereby confirming the RHAMM(low) nature of these cells and supporting the lack of effects of the antibody treatment. Correspondingly, the cells preserved an elongated/stretched morphology.(b)Curiously, anti-CD44 clearly bound (and immobilized) uAD-MSCs did not appear to cause any significant morphological alteration to them. This would appear to indicate that the anti-CD44 effect on these cells may be due to specific signaling rather than to a decreased adhesion to substrate.(c)The RHAMM(high) cell types showed similar effects upon anti-RHAMM and anti-CD44 treatment: the elongated/stretched phenotype was lost and the cells acquired a more round morphology. It is noteworthy, however, that the two antibodies had virtually identical effects on the motility of the two cells, but dAD-MSCs were round already after 1 h of exposure, while aSCs required a longer time (effect apparent at 24 h); due to the different kinetics of the reduction in adhesion, also in this case, we are tempted to ascribe the effects on motility to a signaling cascade triggered by the receptor binding rather than to loss of adhesion.Motility necessarily requires cells to adhere to a substrate; it can be argued that the antibodies may affect the first by reducing the second, which may cause cells to adopt a rounder phenotype. We investigated morphological effects through a tubulin stain, while at the same time, highlighting the antibody binding by using fluorescently labelled anti-RHAMM and anti-CD44 B:(a)AntiThe above observations therefore confirm the RHAMM-dependency of both motility and morphology of dAD-MSCs, similarly to SCs and differently from uAD-MSCs. Our data also suggest that RHAMM and CD44 use complementary circuits to affect motility, with RHAMM possibly more critical than CD44 for Schwann(-like) cells. Last, the role of both receptors on motility may not be directly linked to them being involved in the adhesion to the substrate, although it must be emphasized that this result strictly refers to poly(D-lysine)-coated plastics.We have demonstrated that RHAMM expression is high in Schwann cells and it is also a peculiarity of the Schwann-like differentiation of AD-MSCs. Further, we have shown that RHAMM is critical for the motility of these RHAMM(high) cells, whereas CD44 is apparently more critical for that of the RHAMM(low) uAD-MSCs; while it is clear that both HA receptors have key roles in cell motility, our data appear to support literature data (not on PNS cells) that their stimulation is complementary and has cell-dependent effects. The results of this study offer some important indications in the context of a peripheral nerve regenerative therapy: Firstly, that the motility also of freshly differentiated Schwann-like cells can be boosted by some form of supplementation with HA; secondly, that attention must be paid to develop tools  that can maintain a correct RHAMM(high) phenotype and a continuous stimulation of this receptor.


The COVID-19 viral infection, caused by the novel coronavirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is a currently ongoing global pandemic that, as of mid-October, 2020, has resulted in more than 38.7 million confirmed cases globally and has caused more than 1.1 million fatalities. COVID-19 infection is associated with severe life threatening respiratory and cardiac complications such as acute respiratory distress syndrome (ARDS), pneumonia, shock, cardiac arrhythmias, myocardial infarction and heart failure, particularly in the acute infectious stage. Acute myopericarditis is another reported cardiac complication of COVID-19. Case reports have been limited in reporting the effects of COVID-19 in the post-symptomatic period.In this article, we present a case of acute myopericarditis resulting 6 to 8 weeks after testing positive for COVID-19. Here we will breakdown the initial emergency department (ED) presentation, with particular attention to the electrocardiogram (ECG) findings of acute myopericarditis. This case, to the our best knowledge and after an extensive literature review, depicts the first case of myopericarditis in the post COVID-19 infection recovery phase. The COVID-19 viral infection, caused by the novel coronavirus SARS-CoV-2, is an ongoing global pandemic that has resulted in more than 38.7 million confirmed cases globally and has caused more than 1.1 million deaths as of mid October 2020. The United States of America has the highest number of reported cases and deaths worldwide, with more than 8 million confirmed cases and 250,000 deaths as a result of the virus, according to the World Health Organization (WHO) statistics ..14].It is seen more prominently in the inferior and lateral leads. In the rightward-pointing axis leads aVF and v1, reciprocal PR segment elevation figure  can be sIn addition, Spodick’s sign may also serve as an important distinguishing electrocardiographic tool between the acute pericarditis and acute coronary syndrome. In our case, the presence of PR segment depression as well as reciprocal PR segment elevation, in addition to Spodick’s sign, made the diagnosis of acute myopericarditis highly probable. Limitations of this case report include that we did not obtain histological confirmation of myocarditis and/or isolation of the SARS-CoV2 viral genome in the myocardium. For this we would have needed an endomyocardial biopsy, and such an invasive procedure was not indicated in this case.The diagnosis of acute myopericarditis should always be considered when a patient presents with chest pain and suspected STEMI. There are many cardiotropic viruses that are well known to cause pericardial and myocardial injury such as influenza virus, enterovirus, or parvovirus and now SARS-CoV-2. When considering myopericarditis, the clinician should always consider a viral etiology, especially in the post-viral infection period. Meticulous analysis of the 12 lead ECG in suspected myopericarditis, placing particular attention to the ECG findings mentioned above is important in order to avoid delays in diagnosis and treatment, including exposing the patient to an unnecessary cardiac catheterization. Nonetheless, the false-positive cardiac catheterization laboratory activation for a suspected STEMI is relatively common in clinical practice, especially in the era of emphasis on rapid time to reperfusion therapy metrics .This case report describes myopericarditis in a patient that has recovered from COVID-19 pneumonia several weeks prior. This case report contributes to the currently evolving COVID-19 literature and describes yet another complication of this infection, and our evolving understanding of it.


The pandemic caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has led to several concerns on male fertility. Nowadays, there are numerous unanswered questions, for example: is the virus present or not in the seminal fluid of infected subjects? Could the seminal fluid represent a way of sexual transmission for the virus? Why do men appear to be more susceptible than women? Several studies have been carried out to ascertain the presence of SARS−CoV−2 in the seminal fluid, with contrasting results; the expression of angiotensin-converting enzyme-2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) in the testes and in the male genital tract led to speculation about the possible presence of the virus in the seminal fluid. However, it was found that ACE2 and TMPRSS2, used by the virus to enter host cells, are expressed differently in certain testicle cells , yet the testicle cells in which ACE2 and TMPRSS2 molecules are simultaneously expressed are rare. This fact would suggest that the virus is not able to enter testicular cells, that it is not present in the seminal fluid and that it cannot infect male germ cells. However, the direct influence of SARS-CoV-2 on the testes is still to be evaluated, and recent results are very controversial. SARS-CoV-2 could enter the testicle using alternative paths and lead to alterations in testicular functionality. Another plausible consideration is that the COVID-19 disease could also indirectly cause alterations to testicular activity, since the fever and the cytokinic storm generated by the immune system can lead to damage of the testicular activity, consequently compromising male fertility. Although the literature provides controversial evidence, the purpose of this review is to lend a general overview about the state of the art. Despite the lack of studies, it would represent a starting point for further investigation about the effect of this coronavirus on male fertility. Multiple cases of severe pneumonia provoked by a β coronavirus termed Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) were reported in Wuhan, China, in December 2019 . Since tIn fact, coronaviruses have already been identified in several hosts such as bats, mice, cats, dogs, bulbuls, camels, and other mammals. Most of the coronaviruses that affect humans are associated with fairly mild respiratory symptoms  but SARSCoronaviruses are a family of large, single-stranded, enveloped RNA viruses. The RNA-viral genome is contained in a nucleocapsid, which itself is located within a viral envelope . The envTo enter human cells, SARS-CoV-2 uses angiotensin-converting enzyme-2 (ACE2) as a key receptor. ACE2 is located in different body organs such as kidneys, heart, intestines, liver, lungs, and testes; therefore, cells which have a high expression of ACE2 could be a potential target of the virus. ACE2 is a transmembranal zinc metallopeptidase with a high homology to the classic ACE. ACE isoforms are part of the renin–angiotensin–aldosterone system (RAAS) that plays a crucial role in the regulation of blood pressure and fluid balance. ACE converts Angiotensin I into Angiotensin II (Ang II), whereas ACE2 converts Angiotensin II into Angiotensin 1-7. While Ang II could have dangerous effects on the kidneys, heart, and lungs, the Angiotensin 1-7-Mas receptor axis plays a healthy role because it has vasodilatory, anti-inflammatory, and anti-fibrotic actions ; so evenThe extracellular domain of ACE2 is a cell surface receptor for the Spikes glycoproteins (S domain) on the SARS-CoV-2 envelope. In order to enter host cells, SARS-CoV-2 uses ACE2 and host proteases such as a transmembrane serine protease 2 (TMPRSS2) that cleaves and induces a conformational change to the viral S domain, allowing the fusion of the viral and host membranes . In the KE.In fact, as ACE2 is highly expressed in human testes, it is relevant to evaluate whether COVID-19 in males, via ACE2, might damage fertility. ACE2 is expressed in the testes and epididymis, in particular in Leydig cells, Sertoli cells, and spermatogonia . MoreoveAccording to this study, ACE2 is expressed in male myoid cells, spermatogonial stem cells, and Leydig cells, while TMPRSS2 expression is detected predominantly in elongated spermatides. Despite the fact that a small proportion of spermatogonial stem cells express ACE2 and TMPRSS2, finding cells which co-express both genes is extremely rare, and scRNAseq data results suggest that spermatozoa may not be susceptible to virus infection owing to the lack of ACE2 and TMPRSS2 co-expression. Nevertheless, we have to consider that the virus could be present in the seminal fluid, since this not only contains spermatozoa but also round cells  co-expressing ACE2 and TMPRSS2. Furthermore, since semen is also formed by prostatic components, it would be appropriate to better determine which prostatic cells co-express ACE2 and TMPRSS2. For instance, it is claimed that TMPRSS2 is widely expressed in the prostate epithelial cells, including the apical plasma membrane of the prostate luminal cells and it is also released into seminal fluid as a component of prostasomes, organelle-like vesicles .https://www.proteinatlas.org/humanproteome/sars-cov-2 shows in which male reproductive organs SARS-CoV-2 related proteins are simultaneously expressed, and since, in seminal vesicles, these proteins are simultaneously expressed, virus could be present in the seminal fluid, and semen could be one of the pathways of viral transmission  could modulate testosterone secretion .Although the testicular expression of ACE2 could point to the opinion that the virus might possibly enter into the testicles, not all literature is concordant.In fact, https://www.proteinatlas.org/humanproteome/sars-cov-2  and can survive in the male reproductive system because the testicular immune response is related to sperm survival . Moreovers-cov-2 .However, the risk of a viral contamination due to genital fluids, even with low rate, is not unexpected. Recently, many different studies have demonstrated that men suffer worse clinical outcomes and COVID deaths than women. A variety of factors may cause disparities in sex-specific disease outcomes. For instance, sex-specific differences could be related to steroids and X-linked genes; it is well known that men and women differ in both innate and adaptive immune responses . Sex-speTLR7 is expressed by innate immunity cells; it recognizes viral RNA, promoting the production of antibodies and inflammatory cytokines such as IL6. Nevertheless, in women, the production of IL6 is lower than in men, and it is linked to greater female longevity .Genetic and hormonal differences may give rise to higher male susceptibility. Although the etiology of COVID-19 is probably multifactorial , one of the reasons for this different gender vulnerability could also be related to the production of androgens. According to La Vignera et al., androgens sustain the expression of ACE2; this fact may explain the reasons why men are more exposed to the virus. In fact, prostate cancer patients undergoing Androgen Deprivation Therapy (ADT) show a lower expression of ACE2; therefore, ADT could deter SARS-CoV-2 penetration . EventuaThe pandemic due to SARS-CoV-2 has generated increasing concerns about male fertility and reproduction. The evidences in the literature are very controversial, and further studies are needed to evaluate if the virus can damage male reproductive capability. It is argued that testicular cells expressing both ACE2 and TMPRRS2 are rare, and this fact suggests that the virus may not harm male gametes. However, SARS-CoV-2 could indirectly compromise male gametes, testicular cells, and therefore fertility because the fever and the cytokine storm associated with COVID-19 induce a sperm DNA fragmentation and reduce the male reproductive potential. Finally, although the evidence is controversial, semen could be a viral transmission way. The seminal fluid is formed by several components that not only derive from the testicles but also from the prostate; in fact, it has been suggested that some prostatic cells express TMPRSS2 and release it onto the seminal fluid. This evidence leads to speculation about the fact that semen could represent a SARS-CoV-2 transmission way. The Italian Society of Andrology and Sexual Medicine (SIAMS) stresses the need to carry out further studies aimed at uniquely determining the effects of the virus on testicular functions and detecting its presence in the seminal fluid . In the The evaluation of the presence of SARS-CoV-2 in the seminal fluid is also very important for the semen cryopreservation procedures in liquid nitrogen since viruses stored in liquid nitrogen retain their pathogenic properties . FinallyAN designed the review, carried out an analysis of scientific literature about SARS-CoV-2 and male fertility, and wrote the manuscript. EA evaluated cellular lines in testes and seminal fluid. SC and LA carried out a study concerning spermatozoa and oxidative stress. PL-S analyzed the data and manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fusobacterium nucleatum acting as a keystone colonizer. Microbiome-based machine learning showed high diagnostic and prognostic power for peri-implant diseases and strain-level profiling identified a previously uncharacterized subspecies of F. nucleatum to be particularly associated with disease. Altogether, we associated the plaque microbiome with peri-implant diseases and identified microbial signatures of disease severity.Dental implants are installed in an increasing number of patients. Mucositis and peri-implantitis are common microbial–biofilm-associated diseases affecting the tissues that surround the dental implant and are a major medical and socioeconomic burden. By metagenomic sequencing of the plaque microbiome in different peri-implant health and disease conditions , we found microbial signatures for peri-implantitis and mucositis and defined the peri-implantitis-related complex (PiRC) composed by the 7 most discriminative bacteria. The peri-implantitis microbiome is site specific as contralateral healthy sites resembled more the microbiome of healthy implants, while mucositis was specifically enriched for  Approximately 12 million implants are placed every year worldwide6, but the past 3 decades have seen the emergence of two new oral diseases: peri-implantitis, which affects both the soft and hard tissues surrounding the implant, and mucositis, which precedes peri-implantitis and involves instead only the soft tissues11. Mucositis affects >50% of the implants, while almost 20% of implants develop peri-implantitis16. About half of all inserted dental implants are thus suffering from diseases, leading in most cases to implant loosening or to the need for implant removal18 with very large clinical and socio-economical burdens and serious impairment of patients’ quality of life22.Since the late 70s, the use of dental implants to replace missing teeth has become an increasingly common clinical practice and continuous technological innovations have now made implant therapy more reliable and accessible to the population16. The disease is the result of a continuum of inflammation, tissue destruction, and microbial pressure. Similarly to what is known for periodontal diseases, these factors are influenced also by host-specific immune-mediated response and genetics and are partially modulated by lifestyle and environmental factors23. Despite significant advances made in both periodontal microbiology and pathobiology, it is still unsettled whether the primary disease trigger is the microbial challenge or the hyperinflammatory state itself24. However, differently from the case of bacteria associated with periodontal diseases that have been studied for decades, the only recent emergence of peri-implant diseases limited similar investigations for mucositis and peri-implantitis. Although some known periodontitis-associated bacteria may also be connected with peri-implant diseases, different microorganisms have been suggested to be involved in these two clinically distinct conditions29. A thorough profiling of the microbiome associated with peri-implant diseases is thus an important step to undertake to then help better contextualize host response, genetics and environmental factors, and start moving toward the development of diagnostic, preventive, and therapeutic approaches.Peri-implant diseases are clinically characterized by soft tissue inflammation (mucositis), which may lead to loss of supporting bone (peri-implantitis)30 to profile the plaque microbiome56. Compared to the shotgun metagenomic approach59, this cost-effective technique is unable to identify most microbes at the species level and cannot profile the overall functional potential of the microbial community. Second, the oral human microbiome61 requires large sample sizes and intra-subject microbiome controls  to account for its large inter-subject variability that are not considered in the available studies56. A recent study sampled multiple implants from the same individual but on a small cohort (n = 18) of also periodontal patients55. Third, mucositis and peri-implantitis are very related diseases that should be considered together, but the only two works in which both conditions were sampled focused specifically on the role of smoking on the plaque microbiome42 or had a very small sample size and no intra-subject controls43. There is thus the need for a high-resolution metagenomic investigation of a large cohort including both mucositis and peri-implantitis and controlling for inter-subject variability.Currently available studies investigating the link between the plaque microbiome and peri-implantitis have, however, important limitations. First, they all rely on the 16S rRNA gene sequencing approachIn this work, we investigated the plaque microbiome associated with peri-implantitis and mucositis in a cohort of 72 patients by sequencing a total of 113 metagenomes to identify specific microbial signatures associated with peri-implant diseases. Our study design included, as controls, both healthy implants and teeth that were sampled from healthy sites from healthy individuals and from the contralateral healthy site with respect to the mucositis or peri-implantitis sites. The analysis included not only the investigation of the quantitative taxonomic composition of the plaque microbiome but we also explored the functional potential of the microbiome and the specific strains populating these plaque microbial communities.57 of the plaque microbiome of 113 samples from individuals with dental implants. We profiled subjects with only healthy implants , at least an implant with mucositis , and at least an implant with peri-implantitis  (Table 8 (see “Methods”).In order to study the role of the oral microbiome in peri-implant diseases (mucositis and peri-implantitis), we performed shotgun metagenomic sequencing1) Table  and S2. 11 bases , BOP, SUP, and peri-implant bone loss (see “Methods”). The three clinical conditions were not confounded by other factors such as demographic, anamnestic, clinical, and implant-related characteristics Table  and S2. 64 (see “Methods”), highlighted a clear distinction between the plaque from sites with peri-implantitis and from healthy implants in healthy individuals . The overall functional potential of the microbial communities of the plaque assessed using HUMAnN265 (see “Methods”) was also clearly distinctive for the two conditions  at the level of abundance of single-gene families . This trend points at a plaque microbiome in peri-implantitis composed by fewer dominant species that are encoding a smaller number of functions. The healthy plaque was not only richer but also more variable among individuals . At the whole-microbiome level, peri-implantitis is thus associated with a relatively low diversity microbiome with a well-defined and inter-patient conserved set of bacterial taxa.Analysis of the quantitative taxonomic composition of the plaque microbiome via MetaPhlAn2ies Fig.  as well ies Fig. . The twoies Fig. , althougals Fig. , while dp value < 0.05) between the disease and healthy state, with 54 of them passing multiple hypothesis testing correction  q < 0.1, Supplementary Data Tannerella forsythia to 52% of Treponema lecithinolyticum. The same species had lower but not negligible prevalence in the control sites with T. forsythia, Treponema socranskii, and Fusobacterium nucleatum exceeding 50% prevalence in healthy conditions  and Selenomonas  species characterize the complex and diverse healthy plaque. Most healthy-associated species belonged to the genera Actinomyces (7 species), Capnocytophaga (4 species), Neisseria (4 species), Rothia (3 species), and Streptococcus (5 species) that have no species associated with peri-implantitis.The taxonomic composition of the peri-implant microbiome was characterized by 71 species that were differentially abundant  and again very clearly differentiating the two conditions Fig.  and S7. 66. Considering the ten most strongly peri-implantitis-associated species , third (T. forsythia), and seventh (Treponema denticola) based on their effect size. This highlighted that P. endodontalis (second in the ranking) and F. fastidiosum (fifth) should be considered in the same class of key species for peri-implantitis. The other three bacteria in the ten strongest peri-implantitis-associated species  are also newly associated with the disease, but because they show a smaller effect size compared to the red complex triad, we considered them comparatively less discriminative. We thus propose to define the PiRC of species strongly characterizing peri-implantitis sites as the set of microbes composed by the red complex triad, by the two newly associated P. endodontalis and F. fastidiosum species, and by the Prevotella intermedia and F. nucleatum species found associated with peri-implantitis at higher effect size than T. denticola, the third bacterium of the red complex triad. The PiRC index defined as the cumulative abundance of the species in the PiRC complex is highly discriminative for the two conditions  and teeth (n = 22) in the same volunteers who were sampled from healthy and diseased implants . We found that the microbiome samples of contralateral healthy sites clustered together with the healthy samples regardless of the disease status of the main sites. Indeed, while as expected healthy main sites and their healthy contralateral sites in subjects without peri-implantitis do not have different microbiomes , the healthy contralateral sites of main peri-implantitis sites do not differ from main healthy sites in volunteers without peri-implantitis  nor from the healthy contralateral sites of healthy sites . Peri-implant sites have instead clearly distinct microbiomes from their healthy contralateral sites , from main sites of healthy subjects , and from contralateral sites of healthy subjects . These findings were confirmed by the analysis of the functional potential of the microbiome  and assessed its structure as compared to healthy individuals and peri-implantitis patients. Quantitative microbiome profiles  and healthy implant control group  irrespective of potential confounding factors including sampling procedures . In particular, P. endodontalis is the greater contributor, being the main responsible for 95 associations (36%), followed by T. forsythia .72 applied in cross-validation (see “Methods”)73, we found that peri-implantitis can be predicted at high accuracy, with an area under the receiver operating characteristic curve (AUC) of 0.91 . Second, using PPD values at 6 months as a proxy for improvement or worsening of the clinical condition, we found that the machine-learning classifier trained in cross-validation on the mucositis microbiome was predictive for positive or negative PPD variation , we found that the PiRC index is correlated with mean PPDs for the mucositis samples . By using a pangenome-based strain profiling tool77, we characterized the dominant F. nucleatum strains in the 113 samples considered in this study as well as in 135 additional plaque samples from an American healthy metagenomic cohort63 and 48 plaque samples from patients with periodontitis62. We recovered a total of 208 F. nucleatum strain profiles that were clustered into five distinct subspecies  than healthy sites. Conversely, F. nucleatum subsp. polymorphum was enriched in healthy subjects and control sites. Interestingly, the newly identified and unexplored F. nucleatum Cluster 5 was significantly associated with peri-implantitis and mucositis samples, when compared to healthy subjects and controls . Three additional periodontitis samples harbored a member of F. nucleatum Cluster 5, and the association of this subspecies with disease status held true even when including samples with periodontitis . Taken together, these results suggest that only a subset of F. nucleatum strains including a newly identified subspecies without cultivated representatives is associated with peri-implant diseases and identifying Fusobacterium subspecies in the clinic may have prognostic relevance.We then tested the association of specific subspecies with the clinical conditions. Strains belonging to 80 and diabetes82. The seven bacterial species we included in the PiRC are potential appealing targets for diagnostic and prognostic assessment of the plaque microbiome and the basis for more targeted mechanistic investigation of the host–microbiome interaction network in implant-associated diseases.In this work, we profiled the microbial composition of the plaque biofilm in implant diseases overcoming in resolution (shotgun metagenomics versus 16S rRNA sequencing), sample size (113 metagenomes), and inter-individual microbiome control  the existing investigations. We found strong taxonomic and functional biomarkers for disease and highlighted how peri-implantitis converges toward a low-diversity well-defined pathogenic set of taxa. Importantly, the link that we characterized between peri-implant disease state and the plaque microbiome is substantially stronger than what was previously observed for other oral conditions that can have a role in peri-implantitis such as smokingF. nucleatum is a particularly relevant member of the PiRC because it appears at an initial stage in the inflammation (mucositis). This species is indeed overly abundant before the onset of peri-implantitis and precedes the stronger microbial shift involving many additional taxa. F. nucleatum is a particularly diverse species, and of its known subspecies, only F. nucleatum subsp. nucleatum is connected with disease states. A new subspecies provisionally named F. nucleatum Cluster 5 is even more associated with peri-implantitis and mucositis and should be the basis for further characterization of this clade.Our data also suggest that F. nucleatum and other taxa of the PiRC might be associated with more or less favorable outcomes. More work is thus needed to correlate the structure of the mucositis-associated microbiome with the progress of the disease state.Mucositis is perhaps the more actionable disease state, and although even larger sample sizes are needed, its intermediary microbiome configuration that appears to be in between the healthy plaque and the peri-implantitis plaque is of primary relevance to better understand the molecular cascade in peri-implant diseases. We indeed found that the improvement of clinical parameters associated with mucositis (such as probing depth) that we surveyed longitudinally can be predicted starting from the microbiome structure at diagnosis. Specific microbiome-based therapeutic choices are currently not available, but the presence and abundance of The high-resolution characterization of the plaque microbiome in peri-implant diseases is the first step toward a more comprehensive understanding of the role of the microbiome in peri-implant diseases. Although the inter-subject diversity of the peri-implantitis microbiome is relatively low, the predominance of different taxa in the PiRC might be useful for disease stratification. Further stratifying the three main disease classes using larger sample sizes for a series of covariates that are relevant for oral health such as diabetes, smoking, alcohol, and previous history of periodontitis will also be important to refine the microbiome-based models. In addition, the strong signature and the site specificity of the microbiome in peri-implantitis suggest personalized medicine perspectives based on the local use of antibiotics or antiseptics. Local rather than systemic administration would allow to target PiRC taxa minimizing the impact on the entire oral ecology. Further studies are thus needed to define microbiome-dependent disease subtypes and test the efficacy of therapeutic strategies on such subtype stratification.This study was approved by the ethics committee of the University of Trento (no. 2015-024) and was conducted in accordance with the guidelines of the World Medical Association Declaration of Helsinki. Male and female patients having dental implants and regular maintenance of their dental implants were recruited from six different private practices in the Province of Trentino  for this study. The study protocol was explained to each subject, and a signed informed consent was obtained. Inclusion criteria involved good general health as evidenced by the medical history, being at least 18 years of age, not fewer than 8 teeth, at least one functioning oral implant restored with crowns or prostheses for at least 1 year, willingness to participate in the study. Exclusion criteria included pregnancy or lactation, human immunodeficiency virus, use of immunosuppressant medications, bisphosphonates, or steroids, use of chlorhexidine mouthwash or gel during the previous 2 weeks, oral prophylactic procedures within the preceding 3 months, intake of systemic antibiotics or probiotics within the past 6 months.8. In detail, peri-implant health was diagnosed when the implant was surrounded by healthy soft tissue as determined by the absence of BOP or SUP and visible/detectable radiographic bone loss. Implants with only clinical signs of inflammation in one or more sites  and absence of radiographic bone loss following functional loading were classified as peri-implant mucositis, whereas implants with the presence in one or more sites of both clinical inflammation and radiographic evidence of >2 mm bone loss since the prosthesis installation  were diagnosed as peri-implantitis.Patients were identified and selected in the different private practices and were included in one of the following study groups according to the state of health of their dental implants: (a) healthy , (b) mucositis , and (c) peri-implantitis . The selection and inclusion of patients in one of the groups was based on radiographic evaluation of the marginal bone level, clinical signs of inflammation, and/or presence of SUP according to the criteria delineated by the Consensus Report on Peri-implant DiseasesSix experienced dentists examined all patients. Before commencing the study, meetings were organized in order to instruct all dentists on a common protocol for the examination, collection, and measurement procedures. Plaque samples were collected by a limited number of volunteers (one for each study) in order to minimize the potential biases in the sampling methodology across the different examiners. Follow-up meetings were organized every 3 months to ensure consistency of the sampling, and statistical tests on the results , implant system used and nature of reconstruction , type of implant retention , radiographic peri-implant bone loss, width of the keratinized mucosa, as well as PPD, plaque index (PI), BOP, and SUP. The latter four parameters were measured in each patient at the buccal, mesial, lingual, and distal sites of the experimental implant  and of a healthy contralateral implant (if present) or tooth. PI, BOP, and SUP were recorded on a binary scale (presence/absence) for each surface and PPD was measured to the nearest millimeter on the scale. In case of mucositis and peri-implantitis, any eventual subsequent therapy was noted. All patients were anonymized in the clinic assigning a unique patient ID to all subjects. All downstream analyses were performed using the anonymous IDs and anonymized metadata. The mapping between patients and IDs were stored and kept uniquely at the clinic with only the responsible of each center allowed to have access to it.In total, we enrolled for this study 80 patients  contributing with one implant per patient . Eight patients, four in the health group and four in the mucositis group, were excluded due to failure in DNA extraction or library preparation for sequencing.N = 24), an implant with mucositis  and an implant with peri-implantitis  were compared for demographic, anamnestic, and clinical characteristics , gender , history of periodontitis , smoking habit , diabetes , number of functioning implants , number of residual teeth , previous peri-implantitis , and frequency of home oral care . Extensive clinical data were registered both for experimental implants and contralateral implants or teeth , BOP , SUP , and bone loss , all these parameters significantly higher in the peri-implantitis than in the peri-implant health group. PI was instead non-significant  among groups  from a single implant and from the healthy contralateral/implant/tooth  for each patient in each study group. If multiple implants with the same tested condition were present in a patient, one single implant was randomly selected for sampling. The selected sites were isolated using cotton rolls to prevent contamination with saliva and gently dried with an air syringe, and supramucosal and supragingival plaque was removed using sterile cotton pellets. Submucosal and subgingival plaque samples were taken from the deepest probing site at each selected implant and tooth with individual sterile titanium Gracey curettes. Technical replicates were sampled only after the interruption of any eventual bleeding in order to avoid contamination of the microbiological sample. Curettes were preferred to sterile paper points according to HMP63 and to avoid potential contaminating bacterial DNA associated with paper points83. After the collection, samples were immediately placed in separate Eppendorf 1.5-mL microcentrifuge tubes  containing sterile SCF-1 buffer solution 84 and frozen at −80 °C for later analysis. Total genomic DNA was isolated using the QIAamp DNA Mini Kit : an additional enzymatic disruption step for complete lysis of Gram-positive and Gram-negative species was performed, following the manufacturer’s protocol. Isolated DNA was stored at −20 °C. Laboratory control extractions were also performed on prepared sample buffer to ascertain any potential contaminants. Each metagenome was first quantified, and when there was sufficient material (>1 ng), libraries were prepared using the Nextera-XT DNA Kit  using the manufacturer’s protocol. Technical replicates were used only for the cases in which the first sampling did not yield enough DNA and the DNA extraction of the second replicate was added to the first replicate. Libraries were sequenced (2 × 100 bp reads) on the Illumina HiSeq-2000 platform. Shotgun metagenomics produced initially a total of 140 samples that were reduced due to insufficient non-human DNA depth to 113 quality-controlled samples for a total of 111 Gb and with at least 10 Mb of non-human reads.Microbiome samples were collected with a non-invasive procedure adopting a sampling protocol based on the one validated and adopted by the Human Microbiome Project (HMP) consortium85 by trimming positions with quality <15, removing low-quality reads , and discarding reads shorter than 90 nt. Human and bacteriophage phiX174 DNA (Illumina spike-in) was then removed using BowTie286 by mapping the reads against the corresponding reference genomes. We used MetaPhlAn 287 with default parameter settings for the taxonomic characterization of the sampled microbial community. HUMAnN288 was used to generate normalized pathway relative abundances and gene family relative abundance tables according to the metagenome gene contents. The fold change of the contributions of the PiRC Bacteria over the non-PiRC bacteria in the peri-implantitis-enriched pathways have been computed based on the HUMANn2 output.The generated raw metagenomes were processed with FastqMcf89 on MetaPhlAn2 taxonomic abundance profiles and on HUMAnN2 pathway relative abundances and gene family relative abundance profiles. Alpha-diversity, beta-diversity, and multi-dimensional scaling (MDS) analysis have been performed via custom python scripts based on Scipy, version 1.2.1, and Scikit-learn, version 0.20.390. PERMANOVA was performed using the Scikit-bio python library, ver. 0.2.3, and 1000 permutations. We used the FDR correction adopting the Benjamini–Hochberg approach implemented in the python library Statsmodel, ver. 0.9.0.We performed biomarker discovery using LefSeFor the computation of microbiome richness we subsampled the reads to one million reads per sample. Beta-diversity was computed using the Bray–Curtis dissimilarity index after log-transformation of the raw relative abundances to minimize the effect of compositionality. The two-tailed Wilcoxon rank-sum test was used for the comparison unless otherwise stated. Metric MDS implementation, which adopts the Scaling by MAjorizing a COmplicated Function (SMACOF) algorithm to minimize the stress function, was used; the number of runs for the SMACOF was set to 4, the maximum number of iterations for each run to 5000, and the relative tolerance with respect to stress at which to declare convergence was set to 1e−09. Linear regression of the mean PPD versus the PiRC was computed and plotted together with 95% confidence intervals .73, applying MetAML72, which is based on the Scikit-Learn implementation (ver. 0.20.3) of random forest91. We used random forest as this method achieved the highest consistency and lowest overfitting on metagenomic datasets over a large set of learning problems in microbiome research as reported elsewhere72 and demonstrated also for the analysis of the link between the gut microbiome and colorectal cancer73. We applied this framework on MetaPhlAn2 species-level abundance profiles, HUMANn2 gene family relative abundances, PPD at the sampling site, mean PPD across the four sites of each implant, and on combinations of these feature sets. The random forest classifier uses 1000 estimators, a maximum number of features to consider when looking for the best split of 30% of the feature set for MetaPhlAn2 and of the square root of the size of the feature set for HUMANn2. The minimum number of samples in each leaf was set to 5. The other hyperparameters were left as the default in Scikit-Learn (ver. 0.20.3). For each run, we trained and tested the model in a 20 times repeated 10-fold stratified cross-validations, evaluating the resulting predictions through the mean AUC. Models considering mucositis samples only have been trained and tested in fivefold cross-validation due to sample size constraints, and the minimum sample per leaf was consistently decreased to 1. In this case, a sample has been considered in the negative class (control), if the total millimeters of PDD surrounding the implant were decreased at 6 months after the baseline. A sample was considered positive if the total PPD millimeters were higher or equal to the baseline at 6 months.We used a machine-learning framework similar to the one we employed in ref. 79 and PanPhlAn77 on the raw reads of the 113 samples considered in this study, plus 183 additional gingival plaque metagenomes from healthy and periodontitis subjects retrieved from publicly available datasets. Additionally, 56 reference genomes of F. nucleatum were retrieved from the NCBI Assembly database92 and incorporated in the strain-level analysis. For the strain-level profiling through StrainPhlAn, reads were preprocessed to remove short sequences (i.e. length <90 nucleotides) and then mapped against the MetaPhlAn2 markers with Bowtie2 v. 2.3.486. StrainPhlAn was applied with the following parameters: —marker_in_clade=0.2, —sample_in_marker=0.2, —N_in_marker=0.8 and —gap_in_sample=0.8. In total, F. nucleatum was detected in 208 out of 296 samples. The phylogenetic maximum likelihood tree was generated on the multiple sequence alignment using Muscle v3.8.42593 and RAxML v.8.1.1594 with the GTRCAT model. The phylogenetic tree was visualized with GraPhlAn v. 1.1.395, using the genome of Fusobacterium hwasookii as an outgroup to root the tree. PanPhlAn was applied using default parameters on the same raw reads and reference genomes mentioned above. We build a custom F. nucleatum PanPhlAn pangenome from the 56 F. nucleatum reference genomes by annotating each genome with Prokka v1.1296 and by clustering annotated genes with Roary v. 3.897. Subspecies-level clusters were obtained by performing hierarchical clustering (“average” method) on the PanPhlAn presence–absence gene family matrix and by cutting at a height of 0.45. Prior to clustering, we removed gene families that were present in less than three samples or that were present in more than all-minus-three samples or reference genomes. The principal coordinates analysis of Fig. Strain-level profiling was performed by running StrainPhlAnFurther information on research design is available in the Supplementary InformationSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Reporting Summary