Virus genomes reveal factors that spread and sustained the Ebola epidemic.

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.

Unique human immune signature of Ebola virus disease in Guinea.

Despite the magnitude of the Ebola virus disease (EVD) outbreak in West Africa, there is still a fundamental lack of knowledge about the pathophysiology of EVD. In particular, very little is known about human immune responses to Ebola virus. Here we evaluate the physiology of the human T cell immune response in EVD patients at the time of admission to the Ebola Treatment Center in Guinea, and longitudinally until discharge or death. Through the use of multiparametric flow cytometry established by the European Mobile Laboratory in the field, we identify an immune signature that is unique in EVD fatalities. Fatal EVD was characterized by a high percentage of CD4(+) and CD8(+) T cells expressing the inhibitory molecules CTLA-4 and PD-1, which correlated with elevated inflammatory markers and high virus load. Conversely, surviving individuals showed significantly lower expression of CTLA-4 and PD-1 as well as lower inflammation, despite comparable overall T cell activation. Concomitant with virus clearance, survivors mounted a robust Ebola-virus-specific T cell response. Our findings suggest that dysregulation of the T cell response is a key component of EVD pathophysiology.

External quality assessment of SARS-CoV-2 serology in European expert laboratories, April 2021.

BackgroundCountries worldwide are focusing to mitigate the ongoing SARS-CoV-2 pandemic by employing public health measures. Laboratories have a key role in the control of SARS-CoV-2 transmission. Serology for SARS-CoV-2 is of critical importance to support diagnosis, define the epidemiological framework and evaluate immune responses to natural infection and vaccine administration.AimThe aim of this study was the assessment of the actual capability among laboratories involved in sero-epidemiological studies on COVID-19 in EU/EEA and EU enlargement countries to detect SARS-CoV-2 antibodies through an external quality assessment (EQA) based on proficiency testing.MethodsThe EQA panels were composed of eight different, pooled human serum samples (all collected in 2020 before the vaccine roll-out), addressing sensitivity and specificity of detection. The panels and two EU human SARS-CoV-2 serological standards were sent to 56 laboratories in 30 countries.ResultsThe overall performance of laboratories within this EQA indicated a robust ability to establish past SARS-CoV-2 infections via detection of anti-SARS-CoV-2 antibodies, with 53 of 55 laboratories using at least one test that characterised all EQA samples correctly. IgM-specific test methods provided most incorrect sample characterisations (24/208), while test methods detecting total immunoglobulin (0/119) and neutralising antibodies (2/230) performed the best. The semiquantitative assays used by the EQA participants also showed a robust performance in relation to the standards.ConclusionOur EQA showed a high capability across European reference laboratories for reliable diagnostics for SARS-CoV-2 antibody responses. Serological tests that provide robust and reliable detection of anti-SARS-CoV-2 antibodies are available.

Hospitalized patients with diarrhea: Rate of Clostridioides difficile infection underdiagnosis and drivers of clinical suspicion.

OBJECTIVES: Clostridioides difficile infection (CDI) represents a challenging issue, with an evolving epidemiology. Main objectives of our study were: to assess the frequency of diarrhea of overall etiology, including CDI, as a cause of hospital admission or occurring during hospital stay;- to determine the rate of underdiagnosis of community-acquired (CA-), health care associated (HCA)- and hospital onset (HO-) CDI, and explore factors associated with its clinical suspicion by physicians. METHODS: A prospective cohort study included all hospitalized patients with diarrhea at two acute-care hospitals. C. difficile (CD) tests were performed on every stool samples, irrespective of the treating physician request. Factors associated with the likelihood of CD test request by physicians were assessed. RESULTS: We enrolled 871 (6%) patients with diarrhea. CD test performed on all diarrheic stool samples was positive in 228 cases (26%); 37, 106, 85 cases of CA- (14%), HCA- (42%) and HO- diarrhea (24%), respectively. Treating physicians did not request CD test in 207 (24%) diarrhea cases. The rate of CDI underdiagnosis was 11% (24/228); it was higher in CA-CDI (27%, 10/37). Logistic regression analysis identified age >65 years (RR 1.1; 95 CI 1.06-1.2) and hospitalizations in the previous 3 months (RR 1.2; 95% CI 1.1-1.3) as independent factors associated with the likelihood of requesting the CD test by the physician. These risk factors differed by epidemiological classification of diarrhea and by hospital. CONCLUSIONS: Our study confirmed the relevance of CDI underdiagnosis and provided new insights in the factors underlying the lack of CDI clinical suspicion.

Postmortem Findings in Italian Patients With COVID-19: A Descriptive Full Autopsy Study of Cases With and Without Comorbidities.

BACKGROUND: Descriptions of the pathological features of coronavirus disease-2019 (COVID-19) caused by the novel zoonotic pathogen severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emanate from tissue biopsies, case reports, and small postmortem studies restricted to the lung and specific organs. Whole-body autopsy studies of COVID-19 patients have been sparse. METHODS: To further define the pathology caused by SARS-CoV-2 across all body organs, we performed autopsies on 22 patients with COVID-19 (18 with comorbidities and 4 without comorbidities) who died at the National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS Hospital, Rome, Italy. Tissues from the lung, heart, liver, kidney, spleen, and bone marrow (but not the brain) were examined. Only lung tissues were subject to transmission electron microscopy. RESULTS: COVID-19 caused multisystem pathology. Pulmonary and cardiovascular involvement were dominant pathological features. Extrapulmonary manifestations included hepatic, kidney, splenic, and bone marrow involvement, and microvascular injury and thrombosis were also detected. These findings were similar in patients with or without preexisting medical comorbidities. CONCLUSIONS: SARS-CoV-2 infection causes multisystem disease and significant pathology in most organs in patients with and without comorbidities.

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

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

Differential diagnosis of illness in patients under investigation for the novel coronavirus (SARS-CoV-2), Italy, February 2020.

A novel coronavirus (SARS-CoV-2) has been identified as the causative pathogen of an ongoing outbreak of respiratory disease, now named COVID-19. Most cases and sustained transmission occurred in China, but travel-associated cases have been reported in other countries, including Europe and Italy. Since the symptoms are similar to other respiratory infections, differential diagnosis in travellers arriving from countries with wide-spread COVID-19 must include other more common infections such as influenza and other respiratory tract diseases.

Assessment of biorisk management systems in high containment laboratories, 18 countries in Europe, 2016 and 2017.

Europe-wide activities to improve biosafety and biosecurity performed within the frameworks of the European Union (EU)-funded Joint Actions EMERGE and QUANDHIP led to the development of an Integrated European Checklist for Laboratory Biorisk Management (ECL).To better understand different approaches shaping biorisk management (BRM) systems on an operational level in high containment laboratories, the ECL was used to map the implementation of BRM in 32 high containment laboratories in 18 countries in Europe. The results suggest that the BRM elements referring to standard microbiological working practices and the handling of infectious material were fulfilled particularly well. The elements safety exercises involving internal and external emergency responders, and appropriate decommissioning plans were not fulfilled particularly well. BRM in Biosafety Level (BSL) 4 laboratories handling Risk Group (RG) 4 viruses appear to vary among each other less than BSL3 laboratories handling RG 3 bacteria. It is important to agree on comparable regulations in Europe as high containment laboratories are indispensable for a safe, quick and effective response to public health threats. As high containment laboratories may also present a public health risk it is crucial to have robust BRM on organisational and operational levels.

Detection of Viral RNA in Tissues following Plasma Clearance from an Ebola Virus Infected Patient.

An unprecedented Ebola virus (EBOV) epidemic occurred in 2013-2016 in West Africa. Over this time the epidemic exponentially grew and moved to Europe and North America, with several imported cases and many Health Care Workers (HCW) infected. Better understanding of EBOV infection patterns in different body compartments is mandatory to develop new countermeasures, as well as to fully comprehend the pathways of human-to-human transmission. We have longitudinally explored the persistence of EBOV-specific negative sense genomic RNA (neg-RNA) and the presence of positive sense RNA (pos-RNA), including both replication intermediate (antigenomic-RNA) and messenger RNA (mRNA) molecules, in the upper and lower respiratory tract, as compared to plasma, in a HCW infected with EBOV in Sierra Leone, who was hospitalized in the high isolation facility of the National Institute for Infectious Diseases "Lazzaro Spallanzani" (INMI), Rome, Italy. We observed persistence of pos-RNA and neg-RNAs in longitudinally collected specimens of the lower respiratory tract, even after viral clearance from plasma, suggesting possible local replication. The purpose of the present study is to enhance the knowledge on the biological features of EBOV that can contribute to the human-to-human transmissibility and to develop effective intervention strategies. However, further investigation is needed in order to better understand the clinical meaning of viral replication and shedding in the respiratory tract.

Laboratory management of Crimean-Congo haemorrhagic fever virus infections: perspectives from two European networks.

BackgroundCrimean-Congo haemorrhagic fever virus (CCHFV) is considered an emerging infectious disease threat in the European Union. Since 2000, the incidence and geographic range of confirmed CCHF cases have markedly increased, following changes in the distribution of its main vector, Hyalomma ticks.AimsTo review scientific literature and collect experts' opinion to analyse relevant aspects of the laboratory management of human CCHF cases and any exposed contacts, as well as identify areas for advancement of international collaborative preparedness and laboratory response plans.MethodsWe conducted a literature review on CCHF molecular diagnostics through an online search. Further, we obtained expert opinions on the key laboratory aspects of CCHF diagnosis. Consulted experts were members of two European projects, EMERGE (Efficient response to highly dangerous and emerging pathogens at EU level) and EVD-LabNet (Emerging Viral Diseases-Expert Laboratory Network).ResultsConsensus was reached on relevant and controversial aspects of CCHF disease with implications for laboratory management of human CCHF cases, including biosafety, diagnostic algorithm and advice to improve lab capabilities. Knowledge on the diffusion of CCHF can be obtained by promoting syndromic approach to infectious diseases diagnosis and by including CCHFV infection in the diagnostic algorithm of severe fevers of unknown origin.ConclusionNo effective vaccine and/or therapeutics are available at present so outbreak response relies on rapid identification and appropriate infection control measures. Frontline hospitals and reference laboratories have a crucial role in the response to a CCHF outbreak, which should integrate laboratory, clinical and public health responses.

Relationship Between Viremia and Specific Organ Damage in Ebola Patients: A Cohort Study.

Background: Pathogenesis of Ebola virus disease remains poorly understood. We used concomitant determination of routine laboratory biomarkers and Ebola viremia to explore the potential role of viral replication in specific organ damage. Methods: We recruited patients with detectable Ebola viremia admitted to the EMERGENCY Organizzazione Non Governativa Organizzazione Non Lucrativa di Utilità Sociale (ONG ONLUS) Ebola Treatment Center in Sierra Leone. Repeated measure of Ebola viremia, alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, creatine phosphokinase (CPK), lactate dehydrogenase (LDH), activated prothrombin time (aPTT), international normalized ratio (INR), creatinine, and blood urea nitrogen (BUN) were recorded. Patients were followed up from admission until death or discharge. Results: One hundred patients (49 survivors and 51 nonsurvivors) were included in the analysis. Unadjusted analysis to compare survivors and nonsurvivors provided evidence that all biomarkers were significantly above the normal range and that the extent of these abnormalities was generally higher in nonsurvivors than in survivors. Multivariable mixed-effects models provided strong evidence for a biological gradient (suggestive of a direct role in organ damage) between the viremia levels and either ALT, AST, CPK LDH, aPTT, and INR. In contrast, no direct linear association was found between viremia and either creatinine, BUN, or bilirubin. Conclusions: This study provides evidence to support that Ebola virus may have a direct role in muscular damage and imbalance of the coagulation system. We did not find strong evidence suggestive of a direct role of Ebola virus in kidney damage. The role of the virus in liver damage remains unclear, but our evidence suggests that acute severe liver injury is not a typical feature of Ebola virus disease.

Molecular Characterization of Autochthonous Chikungunya Cluster in Latium Region, Italy.

We report partial molecular characterization of isolates from an autochthonous chikungunya virus cluster in Latium Region. E1 sequences from 3 patients differ substantially from sequences from the 2007 outbreak in Italy and lack the A226V substitution associated with increased viral fitness in the Aedes albopictus mosquito vector.

Genetic Diversity of Mycobacterium tuberculosis Isolates in the Metropolitan Area of Rome.

BACKGROUND: The presence in a geographic area of Mycobacterium tuberculosis (Mtb) strains belonging to different phylogeographic lineages and showing different drug susceptibility patterns may suggest recent transmission, with implications in terms of patient clinical management and disease control. The aim of this study was to carry out a preliminary epidemiological investigation of tuberculosis (TB) cases in Rome. METHODS: A total of 232 Mtb isolates, collected from new or previously treated patients, admitted between 2008 and 2014 at 2 hospital settings in Rome with a diagnosis of TB, were analyzed by spoligotyping and analyzing 24 variable-number tandem repeats (VNTR) mycobacterial interspersed repetitive-unit (MIRU) loci. The SITVIT2 database and the MIRU-VNTRplus web applications were used to identify the strain genotypes and to generate phylogenetic trees. RESULTS: Based on the position on the phylogenetic tree, 97.4% of the strains were associated with 1 of the 7 main lineages. The Euro-American lineage was the most commonly represented (81.9%) within both Italian and foreign-born populations, although all main lineages were present. The highest frequency of drug-resistant strains was found among the East-Asian lineage (Beijing genotype) isolated from foreign-born patients. CONCLUSIONS: Dynamics of TB transmission in Rome indicate recent spread of Mtb strains belonging to phylogeographic lineages and clades usually found in countries and geographic areas with a high incidence of TB, similarly to what is observed in most metropolitan areas in Western Europe. Knowledge from molecular and classical epidemiology provides an important tool for disease control.

Status, quality and specific needs of Ebola virus diagnostic capacity and capability in laboratories of the two European preparedness laboratory networks EMERGE and EVD-LabNet.

From December 2013 to March 2016, West Africa experienced the largest Ebola virus (EBOV) outbreak to date, leading to a European-wide activation of laboratory preparedness and response. At the end of the outbreak, laboratories associated with the two European preparedness networks of expert laboratories EMERGE JA and EVD-LabNet were invited to participate in an assessment of the response of European laboratories to the EBOV outbreak, to identify learning points and training needs to strengthen future outbreak responses. Response aspects assessed included diagnostics, biorisk management and quality assurance. The overall coverage of EBOV diagnostics in the European Union/European Economic Area (EU/EEA) was found to be adequate although some points for quality improvement were identified. These included the need for relevant International Organization for Standardization (ISO) accreditation, the provision of EBOV external quality assessments (EQA) in periods where there is no emergency, facilitating access to controls and knowledge, biorisk management without compromising biosafety and a rapid public health response, and the need for both sustained and contingency funding for preparedness and response activities.

Measles Cases during Ebola Outbreak, West Africa, 2013-2106.

The recent Ebola outbreak in West Africa caused breakdowns in public health systems, which might have caused outbreaks of vaccine-preventable diseases. We tested 80 patients admitted to an Ebola treatment center in Freetown, Sierra Leone, for measles. These patients were negative for Ebola virus. Measles virus IgM was detected in 13 (16%) of the patients.

Fatal Outbreak in Tonkean Macaques Caused by Possibly Novel Orthopoxvirus, Italy, January 2015 1.

In January 2015, during a 3-week period, 12 captive Tonkean macacques at a sanctuary in Italy died. An orthopoxvirus infection was suspected because of negative-staining electron microscopy results. The diagnosis was confirmed by histology, virus isolation, and molecular analysis performed on different organs from all animals. An epidemiologic investigation was unable to define the infection source in the surrounding area. Trapped rodents were negative by virologic testing, but specific IgG was detected in 27.27% of small rodents and 14.28% of rats. An attenuated live vaccine was administered to the susceptible monkey population, and no adverse reactions were observed; a detectable humoral immune response was induced in most of the vaccinated animals. We performed molecular characterization of the orthopoxvirus isolate by next-generation sequencing. According to the phylogenetic analysis of the 9 conserved genes, the virus could be part of a novel clade, lying between cowpox and ectromelia viruses.

Circulation of blaKPC-3-Carrying IncX3 Plasmids among Citrobacter freundii Isolates in an Italian Hospital.

Colonizations due to carbapenem-resistant Enterobacteriaceae (CRE) are a source of antimicrobial resistance transmission in health care settings. Eleven Citrobacter freundii strains producing KPC-3 carbapenemase were isolated from rectal swabs during a 3-year surveillance program. blaKPC-3-carrying plasmids were found to belong to the IncX3 group in 9 of the 11 strains, and complete nucleotide sequences were obtained for 2 of them. Our results highlight the possible role of C. freundii as reservoir of resistance genes.

Prioritization of High Consequence Viruses to Improve European Laboratory Preparedness for Cross-Border Health Threats.

Highly infectious diseases can spread rapidly across borders through travel or trade, and international coordination is essential to a prompt and efficient response by public health laboratories. Therefore, developing strategies to identify priorities for a rational allocation of resources for research and surveillance has been the focus of a large body of research in recent years. This paper describes the activities and the strategy used by a European-wide consortium funded by the European Commission, named EMERGE (Efficient response to highly dangerous and emerging pathogens at EU level), for the selection of high-threat pathogens with cross-border potential that will become the focus of its preparedness activities. The approach used is based on an objective scoring system, a close collaboration with other networks dealing with highly infection diseases, and a diagnostic gaps analysis. The result is a tool that is simple, objective and adaptable, which will be used periodically to re-evaluate activities and priorities, representing a step forward towards a better response to infectious disease emergencies.

Enabling Rapid Response to the 2014-2016 Ebola Epidemic: The Experience and the Results of the National Institute for Infectious Diseases Lazzaro Spallanzani.

The unprecedented epidemic of Ebola virus disease (EVD) in West Africa highlighted the need for stronger systems for disease surveillance, response, and prevention worldwide. Tackling an epidemic event today requires a broader view, not only limited to medical management of the patients, but which also includes heroic efforts by clinicians and public health personnel.Since its foundation in 1936, INMI has been devoted to the prevention, diagnosis and care for infectious diseases. In 2009, INMI became a WHO collaborative center for clinical care, diagnosis, response and training on Highly Infectious Diseases. This paper is aimed to present the activities and the challenging issues encountered by INMI during the 2014-2015 EVD outbreak in terms of preparedness and response to the epidemiological, clinical, diagnostic and research controversial aspects of EVD, both in Italy and in the field.

Dilemmas in Managing Pregnant Women With Ebola: 2 Case Reports.

We report 2 cases of Ebola viral disease (EVD) in pregnant women who survived, initially with intact pregnancies. Respectively 31-32 days after negativation of the maternal blood EVD-polymerase chain reaction (PCR) both patients delivered a stillborn fetus with persistent EVD-PCR amniotic fluid positivity.