# MOOD Project (MONitoring Outbreak events for Disease surveillance in a data science context). Drivers and trends of zoonotic pathogens, antimicrobial resistance and Disease X in Europe.

Claudia Cataldo, Annapaola Rizzoli, Luca Busani

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## To cite this version:

Claudia Cataldo, Annapaola Rizzoli, Luca Busani. MOOD Project (MONitoring Outbreak events for Disease surveillance in a data science context). Drivers and trends of zoonotic pathogens, antimicrobial resistance and Disease X in Europe.. Grant Agreement N°: 874850 - MOOD - H2020-SC1-BHC-2018-2020/H2020-SC1-2019-Single-Stage-RTD, Istituto Superiore di Sanità. 2024, pp.296. hal-04696843

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Public Domain
**MOOD Project**
(Monitoring Outbreak events for Disease surveillance
in a data science context)

**Drivers and trends of zoonotic pathogens, antimicrobial resistance and Disease X in Europe**

Edited by C. Cataldo, A. Rizzoli, L. Busani

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PATOLOGIE
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ISTITUTO SUPERIORE DI SANITÀ

MOOD Project
(MOnitoring Outbreak events
for Disease surveillance in a data science context)

Drivers and trends of zoonotic pathogens,
antimicrobial resistance and Disease X in Europe

Edited by

Claudia Cataldo (a), Annapaola Rizzoli (b),

Luca Busani (a)

(a) Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Roma

(b) Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento

ISSN: 1123-3117 (cartaceo) • 2384-8936 (online)

Rapporti ISTISAN

24/16
Istituto Superiore di Sanità

**MOOD Project (MONitoring Outbreak events for Disease surveillance in a data science context). Drivers and trends of zoonotic pathogens, antimicrobial resistance and Disease X in Europe.**

Edited by Claudia Cataldo, Annapaola Rizzoli, Luca Busani

2024, v, 296 p. Rapporti ISTISAN 24/16 (in Italian)

The project “MONitoring Outbreak events for disease surveillance in a data science context” (MOOD) aims to develop innovative tools and services for the early detection, assessment, and monitoring of current and potential infectious disease threats in Europe in the context of global change. Within the MOOD project, a list of prototype infectious diseases including influenza A, tulaeremia, leptospirosis, chikungunya, dengue, Zika, West Nile, Usutu virus infection, tick-borne encephalitis, Lyme borreliosis and Crimea-Congo hemorrhagic fever was defined. Moreover, antimicrobial resistance and a so called “Disease X”, an unknown but potentially pandemic infection, were included. A comprehensive literature search was performed for each disease following the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) protocol adapted for scoping reviews protocol. The selected articles, complemented with general reviews for descriptive information, were extracted to provide a complete and thorough description of the pathogen and the disease, including environmental and social covariates, epidemiological trends, diagnostics, and prevention tools available. The resulting profiles are available for epidemiologists, risk assessors, and public health officers to support them in understanding and implementing risk assessment and early warning actions.

*Key words:* Zoonoses; One Health; Risk assessment; Epidemiology; Ecology

Istituto Superiore di Sanità

**Progetto MOOD (MONitoring Outbreak events for Disease surveillance in a data science context). Driver e tendenze di patogeni zoonotici, resistenza antimicrobica e “Disease X” in Europa.**

A cura di Claudia Cataldo, Annapaola Rizzoli, Luca Busani

2024, v, 296 p. Rapporti ISTISAN 24/16 (in inglese)

Il progetto MOOD (MONitoring Outbreak events for Disease surveillance in a data science context) ha l'obiettivo di sviluppare strumenti e servizi innovativi per l'individuazione precoce, la valutazione e il monitoraggio di malattie infettive emergenti e riemergenti in Europa nel contesto del cambiamento globale. Nel progetto è definito un elenco di malattie infettive prototipo, tra cui l'influenza A, tularemia, leptospirosi, chikungunya, dengue, Zika, febbri West Nile, Usutu, l'encefalite da zecche, borrelliosi di Lyme e febbre emorragica Crimea Congo. Inoltre, sono stati inclusi i profili di resistenza agli antibiotici e "Disease X", quest'ultimo descrive una malattia sconosciuta e potenzialmente pandemica. Per ognuna è stata eseguita una ricerca della letteratura seguendo il protocollo Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) per le scoping review. Gli articoli selezionati sono stati estratti per fornire una descrizione completa e approfondita dell'agente patogeno e della malattia, comprese le covariate ambientali e sociali, i trend epidemiologici, la diagnostica e gli strumenti di prevenzione disponibili. I profili risultanti sono a disposizione di epidemiologi, valutatori del rischio e responsabili della salute pubblica per aiutarli a comprendere e implementare la valutazione del rischio e le azioni di allerta precoce.

*Parole chiave:* Zoonosi; One Health; Valutazione del rischio; Epidemiologia; Ecologia

*Disclaimer*

The document presents the work performed in the framework of the Horizon 2020 project MOOD. The views and opinions of the authors expressed herein do not necessarily state or reflect those of European Centre for Disease Prevention and Control (ECDC) and World Organization for Animal Health (WOAH). The accuracy of the authors' statistical analysis and the findings they report are not the responsibility of ECDC and WOAH. ECDC and WOAH are not responsible for conclusions or opinions drawn from the data provided. ECDC and WOAH are not responsible for the correctness of the data and for data management, data merging and data collation after provision of the data. ECDC and WOAH shall not be held liable for improper or incorrect use of the data.

This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874850 and is catalogued as MOOD094. The contents of this publication are the sole responsibility of the authors and don't necessarily reflect the views of the European Commission.

Acknowledgement: E4Warning Project is joint funder for models used to develop *Aedes* distribution maps.

Per informazioni su questo documento scrivere a: luca.busani@iss.it; claudia.cataldo@iss.it

Il rapporto è accessibile online dal sito di questo Istituto: www.iss.it.

Citare questo documento come segue:

Cataldo C, Rizzoli A, Busani L (Ed.). MOOD Project (MONitoring Outbreak events for Disease surveillance in a data science context). Drivers and trends of zoonotic pathogens, antimicrobial resistance and Disease X in Europe. Roma: Istituto Superiore di Sanità; 2024. (Rapporti ISTISAN 24/16).

Legale rappresentante dell'Istituto Superiore di Sanità: *Rocco Bellantone*

Registro della Stampa - Tribunale di Roma n. 114 (cartaceo) e n. 115 (online) del 16 maggio 2014

Direttore responsabile della serie: *Antonio Mistretta*

Redazione: *Sandra Salinetti*

La responsabilità dei dati scientifici e tecnici è dei singoli autori, che dichiarano di non avere conflitti di interesse.
Working group “Disease profiles”

The following experts of the working group “Disease profiles” contributed to the document:

<table><tr><td>Daniele ARNOLDI</td><td>Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento (Italy)</td></tr><tr><td>Elena ARSEVSKA</td><td>UMR Animals, Health, Territories, Risks, and Ecosystems (Astre), Department of Biological Systems (Bios), French Agricultural Research and International Cooperation Organization for Development (CIRAD), Campus International de Baillarguet, Montpellier (France)</td></tr><tr><td>Xavier BAILLY</td><td>Université de Lyon, INRAE, VetAgro Sup, UMR EPIA, Marcy l'Etoile; Université Clermont Auvergne, INRAE, VetAgro Sup, UMR EPIA, Saint-Genès-Champanelle (France)</td></tr><tr><td>Maria BELLENGHI</td><td>Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Luca BUSANI</td><td>Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Rosanna CAMMARANO</td><td>Servizio Comunicazione Scientifica, Istituto Superiore di Sanità, Rome (Italy)*</td></tr><tr><td>Claudia CATALDO</td><td>Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Alessandra CIERVO</td><td>Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Francesca DAGOSTIN</td><td>Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento (Italy)</td></tr><tr><td>Simon DELLICOUR</td><td>Department of Microbiology, Immunology and Transplantation, Rega Institute, KU Leuven (Belgium); Spatial Epidemiology Lab (SpELL), Université Libre de Bruxelles (ULB), Brussels (Belgium)</td></tr><tr><td>Marco DI LUCA</td><td>Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Mitra DRAKULOVIC</td><td>Department for Communicable Diseases Prevention and Control, National Public Health Institute "Dr Milan Jovanovic-Batut", Belgrade (Serbia)</td></tr><tr><td>Timothee DUB</td><td>Department of Health Security, Finnish Institute for Health and Welfare, Helsinki (Finland)</td></tr><tr><td>Marzia FACCHINI</td><td>Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Beatriz FERNANDEZ MARTINEZ</td><td>Centro Nacional de Epidemiología, Instituto de Salud Carlos III, Consorcio de Investigación Biomédica en Red Epidemiología y Salud Pública, CIBERESP, Madrid (Spain)</td></tr><tr><td>Claudia FORTUNA</td><td>Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Pachka HAMMAMI</td><td>UMR Animals, Health, Territories, Risks, and Ecosystems (Astre), Department of Biological Systems (Bios), French Agricultural Research and International Cooperation Organization for Development (CIRAD), Campus International de Baillarguet, Montpellier (France)</td></tr><tr><td>Soushieta JAGADESH</td><td>International Society of Infectious Diseases, Boston (MA)(USA)</td></tr><tr><td>Ferran JORI MASSANAS</td><td>Cirad, UMR Astre, Montpellier, University of Montpellier, INRAE, Montpellier (France)</td></tr><tr><td>Isabelle LEBERT</td><td>Université de Lyon, INRAE, VetAgro Sup, UMR EPIA, Marcy l'Etoile; Université Clermont Auvergne, INRAE, VetAgro Sup, UMR EPIA, Saint-Genès-Champanelle (France)</td></tr></table>

<footnote>* Retired</footnote>
<table><tr><td>Henna MÄKELÄ</td><td>Department of Health Security, Finnish Institute for Health and Welfare, Helsinki (Finland)</td></tr><tr><td>Giovanni MARINI</td><td>Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento (Italy)</td></tr><tr><td>Giulia MENCATTELLI</td><td>Centro Ricerca e Innovazione, Fondazione Edmund Mach, Centro Agricultura, Alimenti, Ambiente, Università di Trento, San Michele all'Adige, Trento (Italy), Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise, Teramo (Italy)</td></tr><tr><td>Ewy ORTEGA</td><td>UMR Animals, Health, Territories, Risks, and Ecosystems (Astre), Department of Biological Systems (Bios), French Agricultural Research and International Cooperation Organization for Development (CIRAD), Campus International de Baillarguet, Montpellier; Ministère de l'agriculture et de l'alimentation, Direction générale de l'alimentation (DGAL), Paris, (France)</td></tr><tr><td>Sara PIACENTINI</td><td>Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Scilla PIZZARELLI</td><td>Servizio Comunicazione Scientifica, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Simona PUZELLI</td><td>Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Magalie RENE MARTELLET</td><td>Université de Lyon, INRAE, VetAgro Sup, UMR EPIA, Marcy l'Etoile; Université Clermont Auvergne, INRAE, VetAgro Sup, UMR EPIA, Saint-Genès-Champanelle (France)</td></tr><tr><td>Flavia RICCARDO</td><td>Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Annapaola RIZZOLI</td><td>Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento (Italy)</td></tr><tr><td>Kyla SERRES</td><td>Spatial Epidemiology Lab (SpELL), Université Libre de Bruxelles (ULB), Brussels (Belgium)</td></tr><tr><td>Valentina TAGLIAPIETRA</td><td>Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento (Italy)</td></tr><tr><td>Luciano TOMA</td><td>Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome (Italy)</td></tr><tr><td>Wim VAN BORTEL</td><td>Department of Biomedical Sciences, Outbreak Research Team, Institute of Tropical Medicine, Antwerp (Belgium)</td></tr><tr><td>Esther VAN KLEEF</td><td>Department of Public Health, Institute of Tropical Medicine, Antwerp (Belgium)</td></tr><tr><td>Maria Fernanda VINCENTI-GONZÁLEZ</td><td>Spatial Epidemiology Lab (SpELL), Université Libre de Bruxelles (ULB), Brussels (Belgium)</td></tr><tr><td>William WINT</td><td>Department of Biology, Environmental Research Group Oxford Ltd, Oxford (United Kingdom)</td></tr></table>
## Tick-borne encephalitis

<table><tr><td>Valentina Tagliapietra, Annapaola Rizzoli, Maria Bellenghi, Claudia Cataldo, Francesca Dagostin, Simon Dellicour, Timothee Dub, Henna Mäkelä, Giovanni Marini, Luciano Toma, Maria Fernanda Vincenti González, William Wint, Luca Busani</td><td>166</td></tr></table>

## Tularaemia

<table><tr><td>Maria Bellenghi, Claudia Cataldo, Alessandra Ciervo, Francesca Dagostin, Giovanni Marini, Annapaola Rizzoli, Valentina Tagliapietra, Luca Busani</td><td>204</td></tr></table>

## Usutu virus infection

<table><tr><td>Soushieta Jagadesh, Claudia Cataldo, Francesca Dagostin, Di Luca Marco, Annapaola Rizzoli, William Wint, Luca Busani</td><td>224</td></tr></table>

## West Nile disease

<table><tr><td>Annapaola Rizzoli, Giulia Mencattelli, Daniele Arnoldi, Maria Bellenghi, Claudia Cataldo, Francesca Dagostin, Di Luca Marco, Mitra Drakulovic, Soushieta Jagadesh, Giovanni Marini, Beatriz Fernandez Martinez, Valentina Tagliapietra, William Wint, Luca Busani</td><td>245</td></tr></table>

## Zika

<table><tr><td>Maria Bellenghi, Pachka Hammami, Elena Arsevska, Claudia Cataldo, Francesca Dagostin, Marco Di Luca, Claudia Fortuna, William Wint, Busani Luca</td><td>282</td></tr></table>
ACRONYMS

ADIS Animal Disease Information System

ADNS Animal Disease Notification System

AIVs Avian Influenza Viruses

AMR Antimicrobial Resistance

CAESAR Central Asian and European Surveillance of Antimicrobial Resistance network

CCHF Crimean Congo Haemorrhagic Fever

CDC Centers for Disease Control and Prevention

CESME National Reference Centre for Foreign Animal Diseases

CHIK Chikungunya

CHIKV Chikungunya Virus

DENV Dengue Virus

EARS-Net European Antimicrobial Resistance Surveillance Network

ECDC European Centre for Disease Prevention and Control

EFSA European Food Safety Authority

EIV Equine influenza Virus

ESVAC European Surveillance of Veterinary Antimicrobial Consumption

EU European Union

EU/EAA European Union and European Economic Area

HP Highly Pathogenic

HPAI Highly Pathogenic Avian Influenza

IAV-S Influenza A Viruses of Swine

IAVs Influenza A Viruses

LP Low Pathogenic

MS Members States

OECD Organisation for Economic Co-operation and Development

TBE Tick-borne encephalitis

TESSy The European Surveillance System of Infectious Diseases

USUV Usutu virus

VMPs Veterinary Medicinal Products

WAHIS World Animal Health Information System

WNV West Nile Virus

WOAH World Organization for Animal Health

ZIKV Zika Virus
<figure><img src="image_4.png" /></figure>
# INTRODUCTION

Claudia Cataldo, Luca Busani

Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome

The Horizon 2020 project "MONitoring Outbreak events for Disease surveillance in a data science context" (MOOD), financed by the European Commission (Grant Agreement MOOD N° 874850), aims at the development of innovative tools and services for the early detection, assessment, and monitoring of current and potential infectious diseases threats in Europe in a context of global change.

Data processing methods and spatial or mechanistic modelling within the MOOD project integrates epidemiological and genetic data with environmental and socio-economic covariates in an inter-sectorial, interdisciplinary, One health approach. This project is attentive to any link between the risk of disease emergence and biological/sociological characteristics of both women and men.

The disease intelligence within the MOOD project is working to provide indicators, proxies and define "signals" of disease emergence. It will also provide data favouring open/accessible data sources to improve indicator, event-based surveillance, and early detection of emerging infectious threats. The project ensures that, at the European level, the results are realistic, relevant, and valid with regard to the state of disease knowledge and will create a community of experts. Disease profiles and indicators were defined for major airborne, vector-borne, foodborne, and waterborne infectious diseases, including an unknown disease "Disease X" and antimicrobial resistance.

The starting point for disease profile production has been searching for scientific and technical information for the diseases considered in the MOOD project. A multi-step approach has been followed in addressing the search:

1) description of specific objectives for each profile;

2) use of appropriate bibliographic databases;

3) definition of a common standard protocol for the bibliographic search.

A specific focus has been on social roles and behaviours potentially associated with specific disease risks, considering the gender-based differences identified from the literature.

The list of prototype significant infectious diseases that are considered in the MOOD project included influenza A for airborne pathogens, tularaemia and leptospirosis as models of neglected endemic pathogens with multiple transmission routes and reservoirs, chikungunya, dengue and Zika viruses as models of exotic pathogens transmitted by invasive mosquito species, West Nile and Usutu viruses infections as examples of exotic pathogens transmitted by endemic vectors and tick-borne encephalitis and Lyme borreliosis as models of endemic pathogens transmitted by endemic tick vectors. Moreover, antimicrobial resistance, Crimea Congo haemorrhagic fever, and a so called "Disease X", an unknown but potentially pandemic infection, were included.

Each prototype disease was profiled based on information collected from literature review and expert input. A broad approach based on the "Scoping reviews" scheme was adopted to collect and synthesize the available knowledge.

This document collects the disease profiles produced within the MOOD framework elaborated by the working group "Disease profiles", summarizing the available knowledge on several specific topics for each disease or pathogen defined.
The aim of this collection of disease profiles is to provide to risk assessors and public health
officers a general One Health overview of the diseases, with historical trends and description of
the drivers (related to human, animals, ecosystems, and environment) that modulate the impact
of the diseases and should be considered for risk assessments.
METHODOLOGY

Claudia Cataldo (a), Scilla Pizzarelli (b), Rosanna Cammarano (b), Maria Bellenghi (a), Francesca Dagostin (c), Giovanni Marini (c), Annapaola Rizzoli (c), Valentina Tagliapietra (c), Luca Busani (a)

(a) Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome

(b) Servizio Comunicazione Scientifica, Istituto Superiore di Sanità, Rome

(c) Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento

The prototype infectious diseases proposed by the experts of the working group “Disease Profiles” of the project MOOD (“MOnitoring Outbreak events for disease surveillance in a data science context”) are:

- influenza A for airborne pathogens with multiple host species;

- tularaemia and leptospirosis as models of neglected endemic pathogens with multiple transmission routes and reservoirs;

- chikungunya, dengue and Zika viruses as models of exotic pathogens transmitted by invasive mosquito species;

- West Nile and Usutu virus infections as examples of exotic pathogens transmitted by endemic vectors;

- tick-borne encephalitis and Lyme borreliosis as models of endemic pathogens transmitted by endemic tick vectors.

Moreover, antimicrobial resistance, Crimea Congo haemorrhagic fever, and a “Disease X”, an unknown but potentially pandemic infection, were included.

The information was obtained from the literature by conducting a series of scoping reviews, one for each disease, in accordance with a standardized methodology to ensure the quality and repeatability of the process, in accordance with the guidelines provided by Tricco *et al*. (1). This was done in order to achieve a thorough understanding of the factors influencing the ecology, distribution, and trends in Europe of the prototype infectious diseases included in the MOOD project.

The key questions were harmonized, and a list of relevant keywords organized in a standardized search strategy was arranged and launched across the following databases: MEDLINE, Embase, BIOSIS, SciSearch, CABA, Scopus.

Slight modifications to the standardized protocol that simplified the research strategy and took into account disease-specific aspects were applied for scoping reviews on Crimean Congo Haemorrhagic fever, antimicrobial resistance, and a “Disease X”.

Inclusion and exclusion criteria were defined considering study design, language (English or other EU languages), time frame (10 years for epidemiological and pathogen data, 30 years for environmental data), geographical location (Europe), and publication type.

Studies without data or with non-original or duplicated data (reviews, editorials, letters, modelling studies with no data), lacking denominators or reference populations, unavailable full texts, referring to data older than 2000 or gathered outside Europe, were excluded.

The final time frame covered a period from 2000 to 2022.

The results of the literature searches for each prototype pathogen were uploaded in Rayyan (2) to select and label the articles according to the main topics of interest (human, environmental, animal, vector, and reservoirs covariates). The articles of interest were selected screening titles and abstracts. Those relevant were then retrieved full text, and data were extracted using a data
extraction sheet based on the Cochrane Consumers and Communication Review Group's data extraction template. The data extraction results were analysed, providing basic statistics (numbers and frequencies) for each covariate identified.

Selected articles were summarized in the disease profiles, which were prepared by supplementing the literature search with specific bibliography, particularly for general information on pathogens. Each profile is structured as follows:

- biological, ecological and molecular features of the causative agent;

- natural history of disease in humans and vectors, including symptoms, morbidity and mortality;

- availability of preventive, therapeutic and control measures, including licensed or pipeline vaccines;

- epidemiological situation at different spatial scales: past and current trends;

- sociological and demographical dimension affecting susceptibility and exposure, including gender;

- diagnostic procedures and notification systems used at local, national and European scale;

- infrastructure capacity to identify pathogens for each Member State;

- estimated influence of environmental change on the disease future trends.

The original maps and figures were produced using official data provided by the European Centre for Disease Prevention and Control (ECDC) and World Organization for Animal Health (WOAH), and these data were those available at the time of the analysis. Some of the maps were produced in 2024. At the end of the drafting of the disease profiles, the literature search was updated to include the most recent important information on the diseases. The update included major changes in trends and distribution of the diseases, animals and vectors involved. For those diseases that were updated, references for the period 2023-2024 were included.

Specific maps on vectors distribution in Europe that integrated environmental covariates and data were produced in collaboration with E4Warning Project, a joint funder.

## References

1. Tricco AC, Lillie E, Zarin W, O'Brien KK, Colquhoun H, Levac D, Moher D, Peters MDJ, Horsley T, Weeks L, Hempel S, Akl EA, Chang C, McGowan J, Stewart L, Hartling L, Aldcroft A, Wilson MG, Garritty C, Lewin S, Godfrey CM, Macdonald MT, Langlois EV, Soares-Weiser K, Moriarty J, Clifford T, Tunçalp Ö, Straus SE. PRISMA Extension for Scoping Reviews (PRISMA-ScR): Checklist and Explanation. *Ann Intern Med*. 2018 Oct 2;169(7):467-473. doi: 10.7326/M18-0850.

2. Ouzzani M, Hammady H, Fedorowicz Z, Elmagarmid A. Rayyan-a web and mobile app for systematic reviews. *Syst Rev*. 2016 Dec 5;5(1):210. doi: 10.1186/s13643-016-0384-4.
# ANTIMICROBIAL RESISTANCE

Soushieta Jagadesh (a), Claudia Cataldo (b), Annapaola Rizzoli (c), Luca Busani (b)

(a) *International Society of Infectious Diseases, Boston (MA)*

(b) Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome

(c) Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento

## Priority level for EU

AntiMicrobial Resistance (AMR) in bacterial species is a significant public health challenge requiring continuous surveillance efforts globally, as well as in the European Union (EU) and the European Economic Area (EEA). AMR is responsible for over 35,000 deaths every year in the EU/EEA (1).

Twenty-nine EU/EEA countries report cases of pathogens with AMR to the European Antimicrobial Resistance Surveillance Network (EARS-Net), which has collated this data since the late 1990s, and over the previous decade coordinated by the European Centre for Disease Prevention and Control (ECDC) via the European Surveillance System (TESSy), a web-based platform for data submission and storage (1, 2).

In the 29 EU/EEA countries, a joint report from the OECD (Organisation for Economic Co-operation and Development) and the ECDC reported a shift in the overall antibiotic consumption in Europe since 2014, with the average consumption of antibiotics in humans now higher than in food-producing animals, after adjusting for biomass (3). In 2018, 4,264 tonnes of antibiotics were used in humans corresponding to a mean antibiotic consumption of 133 mg of active substance per kg estimated biomass, whereas 6,358 tonnes of antibiotics were used in food-producing animals corresponding to a mean antibiotic consumption of 105 mg per kg estimated biomass.

Overall, consumption was lower in food-producing animals than in humans in 19 of 29 EU/EEA countries. This change in paradigm highlights the efficacy of the measures to reduce the use of antibiotics in food producing animals taken at country-level. The 13th ESVAC report (4) details that, in 2022, sales of antibiotic Veterinary Medicinal Products (VMPs) used for food-producing animals represented 98.4% of total sales in tonnes (Figure 1) and ranged from 2.1 mg/PCU to 254.7 mg/PCU in the 31 participating countries (Figure 2). The total aggregated sales across all reporting countries were 73.9 mg/PCU. The report also demonstrates that in 2020, community consumption of broad-spectrum antibiotics averaged across EU/EEA countries was 3.5 times higher than consumption of narrow-spectrum antibiotics, which should generally be the first-line therapy (3).

As reported in the ECDC "Antimicrobial consumption in the EU/EEA (ESAC-Net) - Annual Epidemiological Report 2021" (later AER 2021), between 2011 and 2020, an increasing trend was observed in this ratio for the EU/EEA overall and for nine individual countries including Bulgaria, Croatia, Estonia, Hungary, Italy, Latvia, Lithuania, Slovakia, and Slovenia. While in eight of the 29 EU/EEA countries including Austria, Belgium, Denmark, Finland, France, Germany, Ireland, and Norway, narrow-spectrum antibiotics consumption was higher in comparison across the same period. In 2021, the EU/EEA population-weighted mean consumption of antibacterials for systemic use in the community was 15 DDD (Defined Daily Doses) per 1000 inhabitants per day, ranging from 7.2 in Austria to 24.3 in Romania (Figure 3) (5).
<figure><img src="image_5.png" /><figcaption>Figure 1. Overall sales, in tonnes, of antibiotic VMPs for food-producing animals in 31 European countries in 2022 – adapted from 13th ESVAC report (4)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Figure 2. Overall sales, in mg/PCU, of antibiotic VMPs for food-producing animals in 31 European countries in 2022 – adapted from 13th ESVAC report (4)</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>Figure 3. Community consumption (DDD per 1000 inhabitants per day) of antibacterials for systemic use among the EU/EEA countries in 2021 – adapted AER 2021 report (5)</figcaption></figure>

The large variability in AMR percentages particularly in the Southern and Eastern countries of the EU/EEA remains a cause for concern (6). In 2017, 7.2% of *Klebsiella pneumoniae* isolates were identified as resistant to carbapenems, with a large variation across EU/EEA from 0% in several countries, such as Croatia, Estonia, Iceland, Luxembourg, Norway, and Slovenia, to 64.7% in Greece. The 14.9% of *Escherichia coli* isolates were identified as resistant to third-generation cephalosporins, ranging from 5.9% in Norway to 41.3% in Bulgaria. Methicillin-resistant *Staphylococcus aureus* (MRSA) invasive isolates reached a proportion of 16.9%, with variations across Europe (1.0% in Norway to 44.4% in Romania). Moreover, the increase of vancomycin resistance from 9% in 2014 to 17% in 2020 in *Enterococcus faecium*, and of resistance carbapenems from 8% in 2014 to 10% in 2020 in *Klebsiella pneumoniae* was also observed (3). Recent research has also reported an increasing 2020 EU/EEA population- weighted mean resistance of 34% to third-generation cephalosporins in *K. pneumoniae*, and 18% and 38% resistance to carbapenems in Pseudomonas aeruginosa and *Acinetobacter* species, respectively (3).

Cassini *et al*. in 2019 (7) measured the health burden of five types of antibiotic-resistant infection (invasive and non-invasive) caused by eight bacteria with 16 resistance patterns in the EU/EAA. The Disability-Adjusted Life-Years (DALYs) estimated were 671,689 (95% Confidence Interval-CI: 583,148-763,966) cases of infections with antibiotic-resistant bacteria in 2015, of which 426,277 (63.5%) were associated with health care. The overall DALY rate is 170 per 100,000 population, which is similar to the combined burden of HIV, influenza, and tuberculosis in the same year in the EU and EEA. The article also identified that the burden was highest in infants (aged <1 year) and people aged 65 years or older, had increased since 2007, and was highest in Italy and Greece.

To date, the AMR percentages for the bacterial species-antimicrobial group combinations under surveillance remains high in the EU/EEA. In 2022, the ECDC estimated the burden of
infections with antibiotic-resistant bacteria under surveillance in the EU/EEA (1). The number of
cases of these infections increased from 685,433 (95% Uncertainty Interval-UI: 589,451-792,873
cases) in 2016 to 865,767 (95% UI: 742,802-1,003,591 cases) in 2019, with a decrease in the
estimate for 2020 to 801 517 (95% UI: 684,955-932 213 cases). These infections resulted in an
estimated annual number of attributable deaths that increased from 30,730 (95% UI: 26,935-
34,836 deaths) in 2016 to 38 710 (95% UI: 34,053-43,748 deaths) in 2019, decreasing slightly to
35 813 (95% UI: 31,395-40,584 deaths) in 2020.

The decrease in the community antibiotic consumption in the year 2020 in Europe is attributed
to the coronavirus disease (COVID-19) pandemic (Figures 4 and 5).

<figure><img src="image_8.png" /><figcaption>Figure 4. Trends of community consumption (DDD per 1000 inhabitants per day) of antibacterials for systemic use among the EU/EEA countries from 2012 to 2021 – adapted from AER 2021 report (5)</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>Figure 5. Trends of hospital sector consumption (DDD per 1000 inhabitants per day) of antibacterials for systemic use among the EU/EEA countries from 2012 to 2021 – adapted from the AER 2021 report (5)</figcaption></figure>
The disruption of routine care in the community due to patient avoidance of health services owing to self-isolation or an inability to access healthcare reduced antibiotic access (8). Moreover, other Non-Pharmaceutical Interventions (NPIs) for COVID-19 limited physical person to person contacts and other infections (9, 10). Studies noted that the changes in antibiotic consumption during the pandemic were less consistent in the hospital sector, with increased consumption of last-line antibiotics, particularly, carbapenems (8). This was observed by the increase in the number of cases of carbapenem-resistant *Acinetobacter* spp. infections in 2020-2021, mostly in countries that had a relatively high percentage of carbapenem-resistant cases pre-pandemic. *Acinetobacter* spp., including carbapenem-resistant isolates, are known to cause outbreaks becoming difficult to eradicate once they become endemic. It is therefore predicted that carbapenem-resistant *Acinetobacter* spp. will continue to expand in the EU/EEA (8).

Distribution of pathogen

AMR surveillance for EARS-net (EU/EEA) and CAESAR (other countries in the European Region of the World Health Organization, WHO) networks focuses on invasive isolates of eight key bacterial species (*Escherichia coli*, *Klebsiella pneumoniae*, *Pseudomonas aeruginosa*, *Acinetobacter* species, *Streptococcus pneumoniae*, *Staphylococcus aureus*, *Enterococcus faecalis* and *Enterococcus faecium*). AMR percentages are presented for a single antimicrobial agent and/or group of antimicrobial agents. Microorganisms with AMR causing notifiable diseases, such as tuberculosis, are also monitored by the WHO Regional Office. The following data is summarized from the “Antimicrobial resistance surveillance in Europe 2023” report (11). In 2021, 11 (25%) of 44 countries reporting data on *S. aureus* had Methicillin Resistant *S. aureus* (MRSA) percentages below 5%. MRSA percentages equal to or above 25% were found in 13 (30%) countries (Figure 6).

<figure><img src="image_10.png" /><figcaption>Figure 6. Invasive *Staphylococcus aureus* isolates (%) resistant to methicillin (MRSA) in 2021 – adapted from “Antimicrobial resistance surveillance in Europe 2023 report” (11)</figcaption></figure>
Two (5%) of 43 countries reporting penicillin non-wild-type *S. pneumoniae* in 2021 had percentages below 5% (Estonia and Latvia), while percentages equal to or above 25% were found in five (12%) countries (Belarus, France, Romania, Serbia and Turkey).

*E. coli* resistance to fluoroquinolones was lowest in northern and western parts of the European Region and highest in Southern and eastern parts in 2021 (Figure 7A). An AMR percentage below 10% was observed in two (7%) of 29 EU/EEA countries (Finland and Norway). Seventeen countries (58.6%) reported a percentage of 25% or above. AMR percentages of 50% or above were observed in neighbouring countries in the European region (North Macedonia, Russia and Turkey) including Cyprus. For third-generation cephalosporin resistance in *E. coli*, 12 (27%) of the countries in Europe reported resistance percentages below 10% (Figure 7B).

<figure><img src="image_11.png" /><figcaption>**Figure 7.** Invasive *Escherichia coli* isolates (%) in EU/EEA countries in 2021 resistant to: fluoroquinolones (ciprofloxacin/levofloxacin/ ofloxacin) (A), third-generation cephalosporins (cefotaxime/ ceftriaxone/ceftazidime) (B), and carbapenems (C), adapted from "Antimicrobial resistance surveillance in Europe 2023 report" (11)</figcaption></figure>

In 2021, AMR percentages below 10% to third-generation cephalosporins were observed for *K. pneumoniae* in seven (16%) of 45 countries in Europe (Austria, Denmark, Finland, Iceland, Norway, Sweden and Switzerland), while 19 (42%), particularly in the Southern and Eastern parts of the Region, reported AMR percentages of 50% or above (Figure 8A). The emergence of carbapenem-resistant *E. coli* was reported in two (7%) of 29 EU/EEA countries (Greece and Cyprus) with percentages of 1% or above in 2021 (Figure 7C). Carbapenem resistance in *K. pneumoniae* was generally low in northern and western parts of Europe with 14 (31%) of 45 countries reporting AMR percentages below 1% (Figure 8B).

Fifteen (33%) countries reported percentages equal to or above 25%, eight of which (18% of 45 countries) reported AMR percentages equal to or above 50% (Belarus, Georgia, Greece, Moldova, Romania, Russia, Serbia and Ukraine).
<figure><img src="image_12.png" /><figcaption>**Figure 8.** Invasive *Klebsiella pneumoniae* isolates (%) in EU/EEA countries in 2021 resistant to: third-generation cephalosporins (cefotaxime/ ceftriaxone/ceftazidime) (A), and to carbapenems (B), adapted from "Antimicrobial resistance surveillance in Europe 2023 report" (11)</figcaption></figure>

While carbapenem resistance in *P. aeruginosa* in 2021, AMR percentages of below 5% were observed in two (5%) of 44 countries reporting data for *P. aeruginosa* (Denmark and Finland), whereas six (14%) countries reported percentages equal to or above 50% (Belarus, Georgia, Moldova, Russia, Serbia and Ukraine) (Figure 9). The percentages of carbapenem-resistant *Acinetobacter* spp. Varied widely, from below 1% in three (7%) of 45 countries reporting data (the Netherlands, Norway and Sweden) to percentages equal to or above 50% in 25 (56%) countries, mostly in Southern and eastern Europe (Figure 10).

<figure><img src="image_13.png" /><figcaption>**Figure 9.** Invasive *Pseudomonas aeruginosa* isolates (%) in EU/EEA countries in 2021, resistant to carbapenems, adapted from "Antimicrobial resistance surveillance in Europe 2023 report" (11)</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>**Figure 10.** Invasive *Acinetobacter* species isolates (%) in EU/EEA countries in 2021, resistant to carbapenems, adapted from "Antimicrobial resistance surveillance in Europe 2023 report" (11)</figcaption></figure>

Resistance to vancomycin in *E. faecium* varied substantially in Europe. In 2021, percentages of below 1% were reported by six (14%) of 44 countries providing data on this microorganism (Finland, France, Luxembourg, the Netherlands, Norway and Sweden). AMR percentages equal to or above 25% were found in 17 (39%) countries, five of which (11% of 44 countries) reported percentages equal to or above 50% (Cyprus, Lithuania, Malta, North Macedonia and Serbia) (Figure 11).

<figure><img src="image_15.png" /><figcaption>**Figure 11.** Invasive *Enterococcus faecium* isolates (%) in EU/EEA countries in 2021, resistant to vancomycin adapted from "Antimicrobial resistance surveillance in Europe 2023 report" (11)</figcaption></figure>
# Drivers of the AMR emergence and spread

The emergence and spread of AMR are driven by a multitude of factors, including human and animal movements, surface water run-off, and exchange of agricultural products (12). Although multisectoral drivers of AMR globally are poorly understood, the strong link between humans and food-producing animals is well-established (13-15). A study across 11 European countries found strong positive correlations of AMR to various antibiotics (ampicillin, aminoglycosides, third-generation cephalosporins, and fluoroquinolones) in *E. coli* isolated from food-producing animals and from humans (16). Systematic reviews have established this association on demonstrating interventions targeting drug consumption in food-producing animals influenced AMR rates in humans and animals (17, 18). Antibiotic consumption is another well-established key driver for AMR, with compelling evidence for dose dependence in humans (19) and animals (20).

Other drivers influencing AMR spread include anthropogenic factors, such as population density (21, 22) and rising incomes (23, 24). Studies show that the rise in Growth Domestic Product (GDP) and living standards in Low and Middle-Income Countries (LMICs) were positively correlated with antibiotic consumption (24, 25) as well as increase in animal protein consumption (26). Studies demonstrate that inadequate local sanitation, pollution of surrounding water bodies from agricultural run-off and release of non-metabolised antibiotics or their residues into the environment through manure/faeces also play a significant role (27-29).

An important anthropogenic driver of AMR is the misuse of antibiotics, including overprescription of antibiotics as well as overuse in livestock. In 30% to 50% of cases, incorrect prescription of antibiotics is attributed to treatment indication, choice of agent, or duration of antibiotic therapy (30, 31). Similarly, use of antibiotics in food animals has been linked to antibiotic-resistant bacteria in humans through contaminated food-derived products, and represents a threat to human health (32-34). Evidence of a proportion of human extra-intestinal extended spectrum cephalosporin-resistant *E. coli* infections originated from livestock was found in observational studies, with poultry as most probable source (35). A study reported that a targeted intervention to reduce the use of antimicrobials in animals resulted in 24% reduction in prevalence of resistant infections in humans (20). Some contradicting evidence reports that animals to human transfer of resistance is negligible (36). However, numerous studies are established that antimicrobial resistant bacteria originating in an animal can be transmitted to humans through the environment, food products, and/or by direct contact (14,33,37,38).

Climate change was found to influence AMR in Europe (39). In particular, temperature is the strongest drivers of bacterial reproduction and can also modulate aspects of horizontal gene transfer. An increasing trend of resistance in *E. coli* and increase of the minimum temperature has been observed, and European countries with 10°C warmer ambient minimum temperatures experienced more rapid resistance increases across all antibiotic classes in *E. coli* and *K. pneumoniae* (40). Another study demonstrated that higher temperatures and humidity increased colonization and infection risk for MRSA (41).

# Natural history of AMR in humans and animals, including morbidity and mortality

## Brief history of the AMR

The first incident of AMR was observed in 1928 with arsphenamine, also known as Salvarsan, an antibiotic introduced at the beginning of the 1910s (42). Sulphonamide resistance was detected
in certain bacteria in 1933 (43). Resistance to penicillin by bacterial β-lactamases was discovered in 1942, several years before the introduction of penicillin as a therapeutic (44). In the case of streptomycin, introduced in 1944 for the treatment of tuberculosis, mutant strains of Mycobacterium tuberculosis resistant to therapeutic concentrations of the antibiotic were found to arise during patient treatment (45). Tetracycline resistance was first observed with Shigella in 1959 (46). Methicillin resistance was identified in the bacteria *Staphylococcus aureus* in 1961 (47, 48), making methicillin resistant *S. aureus* (MRSA) one of the most difficult to treat nosocomial infections. In the mid-1950s horizontal genetic transfer of antibiotic resistance via plasmids was observed in Japan (49). Resistance to cephalosporin ceftazidime was observed in Enterobacteriaceae two years following its introduction in 1955 (50). Vancomycin resistance was identified in the bacteria *Enterococcus* and *S. aureus* in 1989 (51) and 1998 (52), respectively. Transmissible fluoroquinolone resistance was discovered in the late-1990s (27). *M. tuberculosis* strains resistant to four or more of the front-line treatments have appeared and spread rapidly in the late 2000s (53,54), with totally drug resistant strains occurring in the last decade (55). In 2015, AMR was declared a global emergency by the WHO (56).

The first report of antimicrobial usage for livestock was from Britain where Prontosil and other sulphonamides like sulphapyridine were marketed for use in animals from 1938 (57). In 1940, gramicidin was used to treat a mass outbreak of mastitis at New York's World Exhibition (58). The wartime importance of milk production was highlighted by the usage of penicillin for mastitis in both Britain and Denmark in 1943 (59, 60). In 1948, Merck's sulfaquinoxaline was the first antibiotic to be officially licensed for routine inclusion in poultry feeds (57). Antibiotic use also increased in other areas of global food production: sulphonamides were used in commercial bee hives, in aquaculture, and against mastitis in the dairy sector (57). By the 1960s, antibiotics in food production was widespread globally. In France, approximately 30 tonnes were added to animal feed in 1964 (61). In Britain, experts estimated that 41% (84/168 tonnes) of all antibiotics consumed by animals in 1967 were feed additives (62).

Sweden was the first to ban all food animal growth-promoting antibiotics in 1986 (63). Following which, the EU banned avoparcin in 1997 and bacitracin, spiramycin, tylosin and virginiamycin in 1999 (64). The growth promoter bambermycin was banned from all use in EU livestock since 2006. New regulations on veterinary medicines (Regulation (EU) 2019/6) and medicated feed (Regulation (EU) 2019/4) will enter into force within the European Union (EU) from 28 January 2022. The new veterinary Regulation (EU) 2019/6 has extended restrictions for use of some antibiotics in animals to a full ban of certain antibiotics. Around 25 EU/EEA Member States reported reduced antibiotic usage in veterinary medicine, and mainly in animal husbandry, resulting in a 47% decrease in sales of veterinary antimicrobial medicinal products between 2011 and 2021 (65).

## AMR in humans

Overall, in 2021 (11), the most commonly reported bacterial species with AMR in humans was *E. coli* (39.4% of all reported cases), followed by *S. aureus* (22.1%), *K. pneumoniae* (11.9%), *E. faecalis* (8.8%), *E. faecium* (6.2%), *P. aeruginosa* (6.1%), *Acinetobacter* spp. (3.0%) and *S. pneumoniae* (2.5%). Between 2020 and 2021, the number of reported human cases increased for *Acinetobacter* spp. (+43.2%), *E. faecium* (+20.5%) and *E. faecalis* (+14.0%). Minor differences were noted for *S. aureus* (+9.4%), *P. aeruginosa* (+8.2%), *K. pneumoniae* (+8.1%), *S. pneumoniae* (+4.3%), and *E. coli* (+2.8%).

AMR in *K. pneumoniae* is of major concern in the EU/EEA. Recent outbreaks of carbapenemase (NDM-1 and OXA-48)-producing and colistin-resistant *K. pneumoniae* were reported to have a concomitant increase in virulence (66,67), transmissibility and AMR which
pose a considerably higher risk to human health than the *K. pneumoniae* strains that previously circulated.

A recent publication demonstrated that a major part of the increase in reported *Acinetobacter* spp. in 2020-2021, during the COVID-19 pandemic years, consisted of carbapenem-resistant ventilator-associated infections in ICU patients, in the countries with carbapenem resistance percentages in this pathogen exceeding 50% in 2018-2019 with a recent estimate from 2020 attributing 3656 deaths to carbapenem-resistant *Acinetobacter* spp. (68). Multidrug resistant *Acinetobacter* spp. is notoriously difficult to eradicate from the hospital environment once established as these strains can survive on dry surfaces, readily contaminating healthcare providers' hands, and being spread by asymptomatic carriers

Despite the relative decline of MRSA and *S. pneumoniae* percentages in EU/EEA countries, they remain an important pathogen with EU/EEA health burden of MRSA for the period 2016-2020 being the second largest (1).

Vancomycin-resistant *E. faecium* increased from 47124 in 2016 to 117866 in 2020, with a consequent increase in the number of attributable deaths from 1335 to 3414 (1). WHO has listed vancomycin-resistant *E. faecium* as a pathogen of high priority in its global priority list of antibiotic-resistant bacteria, emphasizing the paucity of available and effective treatment options (69).

## AMR in animals

Majority (73%) of all antimicrobials are used in animals for the prevention and treatment of infections (70) and, outside the EU, are also used to improve weight gain and productivity on farms (71). In the report, AMR in animals is divided into three sections: domestic animal husbandry, companion animals, and aquaculture.

### AMR in domestic animal husbandry

Antibiotic use in livestock includes treatment of infections as well as administration of subtherapeutic doses for significant weight gain among the treated animals (72-76). According to Commission Implementing Decision (EU) 2020/1729, which applies from 1 January 2021 until December 2027, monitoring of AMR is mandatory in *Salmonella* spp., *Campylobacter coli*, *Campylobacter jejuni*, and indicator *E. coli*, in the major domestically produced animal populations and their derived meat. In 2021, the population-weighted mean antimicrobial use (AMU) in food-producing animals was lower than in humans in the EU/EEA (3, 77). AMU reduction in chickens was successful in the Netherlands due to the large-scale transition from fast-growing to slow-growing chicken breeds, which required fewer antibiotic treatments (78). Studies report a substantial reduction of AMU on herd level without major impact on pig health economic performance (79, 80).

### AMR in companion animals

Antimicrobial resistance of pet origin poses a major threat to human health, particularly concerning MRSA, other methicillin-resistant staphylococci, vancomycin-resistant enterococci, carbapenemase-producing *enterobacteriaceae* and Extended Spectrum Beta-Lactamase (ESBL) Gram-negative bacteria (81). A study analysing the frequency of methicillin-resistant *Staphylococcus* spp. in pets (82), dogs and cats, highlighted the prevalence of methicillin resistance traits with 40% of the resistant Staphylococcal population identified as *Staphylococcus schleiferi*, 35% *S. aureus*, and 17% *S. intermedius*. The study observed that *S. schleiferi* was more recurrent in dermatitis and ear canal infection, prevalently isolated from dogs similar to resistant
S. intermedius strain. MRSA isolation was associated with deep infections with a similar frequency in dogs and cats. Another study carried out in three Eu countries showed that 19% of the animals received at least one antimicrobial treatment six months preceding sampling and cats and dogs were treated with a standard daily dose of antimicrobials for 1.8 and 3.3 days over one year, respectively (83). The most frequently used antimicrobial was amoxicillin-clavulanate (27%). Resistance of E. coli to at least one antimicrobial agent was found in 27% of the isolates with 18% resistance to ampicillin and 13% multidrug resistant isolates (83).

## AMR in aquaculture

Although aquaculture constitutes only 5.7% of the global antimicrobial use, it carries the highest use intensity per kilogram of biomass, 164.8 mg per kg. Some species of fish, such as catfish, are associated with antimicrobial use rates per kilogram that exceed those in terrestrial animals and humans (84). On the contrary, salmons accounted for the lowest antimicrobial use, although use coefficients in commercial salmon production varied by several orders of magnitude across countries (85-88). A recent study reported seafood as the most common food source of E. coli containing β-lactam resistance genes (89). Moreover, aquaculture settings utilizing antimicrobials serve as reservoirs for AMR genes, providing routes for human and animal exposure to resistant bacteria (90-94). Aquaculture and the aquatic environment have been linked to mobile genetic elements carrying resistance genes of human clinical significance (91, 92, 95). As fish and seafood supply chains are highly globalized (96), it facilitates the distribution of locally generated resistance at a global scale (97, 98). A recent study demonstrated that between 2000 and 2018, AMR in bacteria from aquaculture remained stable (33%) while the resistance from wild-caught aquatic food animals decreased sharply (52% to 22%) (99). The declining resistance observed in bacteria from wild-caught aquatic animals was attributed to reduced human and livestock faecal pollution exposure. Amongst foodborne pathogens, the study observed high rates of resistance to first-line antimicrobial classes and, for Vibrio and Aeromonas spp., moderate to high rates of resistance to antimicrobial classes of last-resort reserved for treatment of multidrug resistant pathogens in humans (99).

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# CHIKUNGUNYA

Claudia Cataldo (a), Elena Arsevska (b), Maria Bellenghi (a), Francesca Dagostin (c), Marco Di Luca (d), Flavia Riccardo (d), Kyla Serres (e), William Wint (f), Luca Busani (a)

(a) Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome

(b) UMR Animals, Health, Territories, Risks, and Ecosystems (Astre), Department of Biological Systems (Bios), French Agricultural Research and International Cooperation Organization for Development (CIRAD), Campus International de Baillarguet, Montpellier

(c) Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento

(d) Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome

(e) Spatial Epidemiology Lab, Université Libre de Bruxelles, Brussels,

(f) Department of Biology, Environmental Research Group Oxford Ltd, Oxford

## Biological, ecological and molecular features of the causative agent

### Disease name

Chikungunya (CHIK).

### Disease agent

#### Common scientific and Latin name

Chikungunya virus (CHIKV) is the etiological agent of chikungunya fever (CHIKF), an arthropod-borne disease transmitted mainly by mosquito species of *Aedes* genus, (*Ae. furcifer-taylori*, *Ae. africanus*, *Ae. luteocephalus*, and *Ae. neoafricanus*, enzootic vectors in African savannah and forest cycles, *Ae. aegypti* and *Ae. albopictus*, vectors in urban areas). The name 'chikungunya' derives from a word in the Kimakonde language, meaning "to become contorted" and refers to the contorted posture of infected patients suffering from severe joint pain (1).

### Taxonomy

CHIKV is an RNA virus that belongs to the Semliki Forest antigenic group of the genus *Alphaviridae* (Family *Togaviridae*), which includes other arthritogenic alphaviruses (as o'nyong-nyong, Ross River, Barmah Forest, and Mayaro viruses) (2). Phylogenetic analyses reveal three genotypes of CHIKV: West African genotype, East/Central/ South African (ECSA) genotype including the Indian Ocean Lineage (IOL), and the Asian genotype (3).

### Disease agent characteristics

CHIKV genome consists of a single 11.8-kbp strand of positive sense RNA, which encodes a 2472 amino acid non-structural and a 1244 amino acid structural polyprotein (4). The polyproteins give rise to the four non-structural proteins (nsP1-4) that make up the viral replication machine, and five structural proteins. Each spherical viral particle is approximately 70 nm in diameter and is comprised of a strand of genomic RNA, encapsulated by capsid (C) proteins, surrounded by a host cell-derived lipid bilayer spiked with heterodimers of envelope proteins E1 and E2 (5). The
other two structural proteins, 6K and E3, are leader peptides for E1 and E2, respectively, and are not observed in abundance in the mature virion (5).

## Physiochemical properties

CHIKV is sensitive to increasing temperatures and can be safely and quickly inactivated when treated at temperatures above 70°C for at least 1 minute. Furthermore, a superior virucidal effect of propanol-based disinfectants over ethanol-based solutions has been demonstrated (6). The efficacy of amotosalen and ultraviolet A light treatment, inactivates high levels of CHIKV has been demonstrated with a log reduction factors LRF >5.0 in both plasma and in platelet components. Additionally, other alphaviruses predicted to be important emerging agents have been successfully inactivated by amotosalen/UVA (7).

## Priority level for EU

CHIKV is currently not endemic in the EU and the majority of the cases are travellers infected during international travel. However, two large outbreaks were reported in Italy in 2007 and 2017, and some autochthonous cases were reported in France in 2010, 2014 and 2017 (source ECDC, European Centre for Disease Prevention and Control). Local transmission in the EU is linked to importation of virus by infected travellers to areas with established presence of competent vectors (*Aedes albopictus* present in many countries in mainland Europe and *Aedes aegypti* in Madeira, Canary Islands, Cyprus and countries around the Black Sea) when environmental conditions favour vector activity. Considering the frequency of travellers between high incidence areas in the world and the European Union and the past experiences in Italy and France, there is a risk of future CHIKF outbreaks in continental Europe. This is why, this disease is prioritized in EU surveillance and the spread of *Ae. albopictus* and other invasive *Aedes* species is monitored in the EU/EEA (European Union/European Economic Area).

## Distribution of the pathogen

CHIKV has been sporadically detected in Africa and Asia and, since 2004, has extended its geographic range causing outbreaks in the Indian Ocean, south-eastern Asia, Europe and the Americas. This global expansion has been possible because CHIKV established a transmission cycle in urban settings using anthropophilic vectors such as *Ae. albopictus* and *Ae. aegypti*.

To date, *Ae. albopictus* has been established as the main CHIKV vector of transmission in Europe. Nevertheless, *Ae. aegypti*, which was eradicated in Europe since the 1950s, has been detected again around the Black Sea in Southern Russia, Abkhazia, and Georgia in 2004 and north-eastern Turkey in 2015 (8) and more recently in Cyprus and the Canary Islands (Ref). In addition, other invasive mosquitoes, *Ae. koreicus* and *Ae. japonicus*, have been introduced and established in several European countries, *Ae. koreicus* is also able to experimentally transmit CHIKV (9). CHIKV outbreaks in Southeast Asia occurred in larger cities, where *Ae. aegypti* mosquitoes were implicated as the primary transmission vector (10).

The presence of competent vectors is the prerequisite for a possible transmission of CHIKV in a given area. When the environmental conditions are favourable, in areas where *Ae. albopictus* is established, viraemic travel-related cases may trigger local transmission of the virus as
demonstrated by the sporadic events of CHIKV transmission since 2007. No events of autochthonous transmission were reported at European level from 2017 to date (February 2023).

Table 1 describes the distribution of *Ae. albopictus* for the years 2017-2022 and in Figure 1 the map of *Ae. albopictus* distribution, as of March 2024, is reported.

<table><caption>Table 1. 5 years monitoring of presence and abundance of *Ae. albopictus* in Europe</caption><thead><tr><th>Country</th><th>2017</th><th>2018</th><th>2019</th><th>2020</th><th>2021</th><th>2022</th><th>CHIKV outbreak</th></tr></thead><tbody><tr><td>Belgium</td><td>A</td><td>A</td><td>A</td><td>I</td><td>I</td><td>I</td><td></td></tr><tr><td>Central France</td><td>I</td><td>I</td><td>I</td><td>E</td><td>E</td><td>E</td><td></td></tr><tr><td>Central Spain</td><td>ND</td><td>ND</td><td>ND</td><td>I</td><td>I</td><td>E</td><td></td></tr><tr><td>Italy</td><td>E</td><td>E</td><td>E</td><td>E</td><td>E</td><td>E</td><td>Emilia-Romagna 2007<br>Lazio and Calabria 2017</td></tr><tr><td>Netherland</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I</td><td></td></tr><tr><td>North France</td><td>A</td><td>A</td><td>A</td><td>A</td><td>A</td><td>A</td><td></td></tr><tr><td>North Germany</td><td>A</td><td>A</td><td>A</td><td>A</td><td>A</td><td>A</td><td></td></tr><tr><td>North Spain</td><td>ND</td><td>ND</td><td>ND</td><td>A**</td><td>A**</td><td>A**</td><td></td></tr><tr><td>Portugal</td><td>A</td><td>A</td><td>A</td><td>A**</td><td>A***</td><td>A***</td><td></td></tr><tr><td>South France</td><td>E</td><td>E*</td><td>E*</td><td>E</td><td>E</td><td>E</td><td>Montpellier 2014<br>Le Cannet-des-Maures 2017</td></tr><tr><td>South Germany</td><td>I</td><td>I</td><td>I</td><td>I</td><td>I****</td><td>I****</td><td></td></tr><tr><td>Spain Mediterranean Coast</td><td>E</td><td>E</td><td>E</td><td>E</td><td>E</td><td>E</td><td></td></tr><tr><td>UK</td><td>A</td><td>A</td><td>A</td><td>A</td><td>A</td><td>A</td><td></td></tr></tbody></table>

A=Absent; ND=No Data; I=Introduced; E=Established; *in expansion; **some plot in the region of Galizia; ***some plot of established *Ae. albopictus* in the south and north of Portugal; ****some plot of established *Ae. albopictus*.

<figure><img src="image_16.png" /><figcaption>Figure 1. Current 1-km probability of presence of *Ae. albopictus* across Europe, produced using random forest and boosted regression trees analyses (source: updated by ERGO for E4Warning Project)</figcaption></figure>
## Ecology and transmission routes

CHIK is a climate sensitive disease, however its transmission is also affected by deforestation, population migration, disorderly occupation of urban areas, and precarious sanitary conditions that favour the amplification of both the virus and the mosquito vectors, and the transmission of the infection (11, 12).

Among factors that probably facilitated recent CHIKV emergence include:

1) availability of immunologically naïve human populations in vast geographic areas;

2) CHIKV reliance on peridomestic and anthropophilic mosquitoes as vectors;

3) increase in international travel,

4) genetic adaptation of CHIKV to a new mosquito vector, *Ae. albopictus*.

CHIKV transmission by *Ae. albopictus* was consistently associated with an aminoacid substitution in the E1 protein (E1-A226V) (13-15). Recently, effective transmission of strains without the A226V mutation was demonstrated in Italy, where *Ae. albopictus* was the implicated vector (16).

## Drivers of the disease emergence and spread

### Ecological drivers

The influence of climate change and variability on infectious disease spread and emergence should, however, not be viewed in isolation as it is expected to interact with other drivers, such as urbanization, land use, and human mobility.

Particularly, the unprecedented increase in human mobility, both at the local and global scales, is considered a major driver for the expansion of dengue and CHIK (17).

Non-human primates are believed to be the primary CHIKV reservoir hosts, and the 5–10-year periodicity of virus activity in a given locality is hypothesized to depend on oscillations in monkey herd immunity. In addition, the virus and/or neutralizing antibodies were detected also in birds, rodents and other small mammals, but their role as reservoir is questionable.

In Table 2 and in Table 3 the most important environmental and vector drivers, which are thought to impact on the spread of CHIKV are reported.

<table><caption>**Table 2.** List of individual CHIKV environmental covariates reported in selected studies, in descending order of importance according to the number of references</caption><thead><tr><td>Environmental drivers</td><td>n. of papers# (n. 3)</td><td>% of impact* (n. 9)</td></tr></thead><tbody><tr><td>Temperature (incubation in experimental condition; average daily temperature)</td><td>4</td><td>44.4%</td></tr><tr><td>Time (season during year)</td><td>4</td><td>44.4%</td></tr><tr><td>Water (rainwater storage tanks)</td><td>1</td><td>11.1%</td></tr></tbody></table>

<footnote># number of papers extracted, the number of references per covariate is higher because more covariates may have been extracted from a document;</footnote>

<footnote>*% calculated on the total number of references.</footnote>
<table><caption>Table 3. List of CHIK vector covariates included in the selected studies in decreasing order of importance according to the number of references</caption><thead><tr><td>Vector drivers</td><td>n. of papers# (n. 12)</td><td>% of impact* (n. 33)</td></tr></thead><tbody><tr><td>Transmission (transmission/infection rate; viral titre)</td><td>14</td><td>42.4%</td></tr><tr><td>Abundance (mosquito density; n. of eggs/ovitrap)</td><td>10</td><td>30.3%</td></tr><tr><td>Activity (transmission rate; biting rate)</td><td>6</td><td>18.2%</td></tr><tr><td>Ecology (n. of breeding sites)</td><td>3</td><td>9%</td></tr></tbody></table>

# Natural history of disease in humans and animals, including symptoms, morbidity and mortality

## Brief history of the pathogen and disease

The earliest report of CHIKF described an outbreak of a dengue-like illness that occurred in 1952 to 1953 in the present region of Tanzania, where an estimated 60% to 80% of the population in this region developed symptoms (1).

Between the 1960s and 1990s, incidental human infection led to numerous, small-scale CHIKV outbreaks in countries throughout Central and Southern Africa, and Senegal, Guinea, and Nigeria in Western Africa (18). In 2004, a large-scale CHIKV epidemic erupted, sweeping down the coast of Kenya into islands on the Indian Ocean (Comoros, Mayotte, Seychelles, Re' union, Madagascar, Sri Lanka, and the Maldives), India, Southeast Asia (Malaysia, Singapore, Thailand), and China (19). Although CHIKV infection in travellers returning to Europe had been reported previously, autochthonous transmission of CHIKV was observed for the first time in Italy in 2007 (20), and in France in 2009 (21). In December 2013, the first cases of locally transmitted CHIKV in the Americas were confirmed in St. Martin, followed rapidly by cases identified throughout the Caribbean and Latin America (22). Currently, CHIKV infection has been reported in different countries on all continents, except Antarctica (23). In some regions, especially in South America, the co-circulation of CHIKV with other arboviruses, such as DENV, ZIKV, Mayaro (MAYV) and yellow fever (YFV), requires rigorous epidemiological surveillance and differential diagnosis strategies (24).

## Disease in humans

Following transmission, CHIKV replicates in the skin and then disseminates to the liver and joints, presumably through the blood (25). The incubation period is 2-4 days and is followed by a sudden onset of clinical disease. Symptoms of CHIKV infection include high fever, rigors, headache, photophobia and a petechial rash or maculopapular rash. In addition, most infected individuals complain of severe joint pain that is often incapacitating (26). Patients often report some stiffness in the distal joints, such as the interphalangeal joints of the hands and feet, ankles, and wrists, particularly upon awakening (27). Myalgia is more frequent in the arms, forearms, thighs, and calves and may compromise the daily activities of patients, particularly when associated with polyarthralgia/polyarthritis. The acute phase of CHIKV infection is usually self-limited and clinical manifestation lasts from a few days to a couple of weeks. The post-acute phase begins after the 21st day of clinical manifestations and continues for three months (28).

<footnote># number of papers extracted, the number of references per covariate is higher because more covariates may have been extracted from a document; * % calculated on the total number of references</footnote>
Only a small proportion of patients remain completely asymptomatic after two-three weeks following the onset of disease. Generally, most patients exhibit only transitory improvements in their clinical condition and relapses occur after a brief "healing" period. Moreover, persistent polyarthralgia or polyarthritis without any change in intensity has been reported by a considerable percentage of patients, which requires analgesic or anti-inflammatory medication to alleviate the pain. During the post-acute phase, the decompensation of pre-existing traumatic or degenerative arthropathies, such as osteoarthritis or tendinitis, occasionally calcified, may occur. Additionally, local manifestations, such as reactional oedema and nerve compression syndromes, particularly of the ulnar, medial, and tibial nerves, which produce cubital, carpal, and tarsal tunnel syndromes, respectively, have also been observed. Morning joint stiffness, neuropathic pain, and peripheral vascular phenomena, such as Raynaud syndrome, have also been described (28). Notably, during this phase, a set of non-specific clinical manifestations that are not always associated with CHIK usually occurs, which may be overlooked by health professionals. The most frequently reported manifestations are chronic fatigue, changes in skin colour (hypo- or hyperchromia), alopecia, decompensated endocrine and metabolic diseases, and decompensation of other pre-existing chronic diseases, such as systemic arterial hypertension, depression, and anxiety (28).

In contrast to the acute phase, the chronic phase of disease has not been extensively investigated. Recurrent joint pain, which can last for years in some cases, is experienced by 30-40% of those infected, although this is not thought to be a result of chronic infection, as infectious virus cannot be isolated from these patients. Radiographic studies are typically normal or show mild swelling, which is consistent with joint pain. It has been suggested that this joint pain is immune mediated. This has not been formally shown, although the presence of autoantibodies has been reported in one case of CHIKV infection with severe musculoskeletal complications (29). Other types of musculoskeletal manifestations may characterise the chronic phase, with the most frequent being tenosynovitis. Typically, two or more tendons are affected, the most common of which are the wrist, finger, and ankle extensors and flexors. Many patients with hypertrophic wrist tenosynovitis complain of nocturnal paraesthesia in the fingers.

During the more recent CHIKV outbreaks, total or partial alopecia on the head or body, predominately in female patients, and ophthalmological alterations, such as uveitis and retinitis, were described during the chronic phase of infection (30).

In new-borns, congenital infections may be accompanied by varying clinical signs, such as fever, lack of appetite, apnoea, skin manifestations, distal and cerebral oedema, encephalitis and haemorrhage (31) (32). Bullous lesions associated to CHIKV infection have also been reported in four-month-old babies, who had 20% of their body surface affected on the second day after the onset of fever (25). Deaths from CHIKV infection is a rare event, but some studies conducted in Brasil described an increased risk of mortality after infection, probably due to neurological affections, mainly in neonates, immunocompromised and elderly (32, 33).

# Availability of preventive, therapeutic and control measures, including licensed or pipeline vaccines

## Therapy in humans

There is presently no licensed targeted therapy for acute CHIKV infection. Treatment is primarily supportive care and includes the use of analgesic and anti-inflammatory medication, rehydration, and rest (34). Going more in depth the most common symptoms, fever and joint pain can be alleviated with the use of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) (35).
## Licensed or pipelined vaccines

In November 2023 the Food and Drugs Administration (United States) approved the first CHIKV vaccine for individuals 18 years of age and older. It contains a live, weakened version of the virus (https://www.fda.gov/news-events/press-announcements/fda-approves-first-vaccine-prevent-disease-caused-chikungunya-virus). Other anti-CHIKV candidates that have been already tested in humans and/or animals include inactivated vaccines, attenuated vaccines, Virus Like Particle (VLP) vaccines, nucleic acid vaccines and chimeric vaccines (36).

## Other prevention measures

Apart from the use of the recently approved vaccine, in the absence of therapeutic strategies, efficient vector control plays a crucial role in CHIKV prevention (37). Integrated anti-virus control is required and should include: a) epidemiological surveillance; b) environmental management focusing on educative actions to eliminate potential mosquito breeding sites and reduce standing water sites; c) chemical control using repellents (mainly for travellers and pregnant women) and insecticides, monitoring the vectors resistance; and d) biological control of eggs, *larvae* and mosquitoes (38) (24) (39) (40). In certain countries such as Germany, Italy, and Spain, tools or apps have been provided to citizens for reporting mosquito presence and biting incidents.

Transmission of CHIKV through transfusion and transplantation has not been reported, but preventive blood safety measures should be considered in case of donors from areas with ongoing viral circulation. Virus prevalence in blood donors ranged from 0 to 2% in areas of reported virus circulation (41, 42).

## Disease specific recommendations

Individuals living in or travelling to endemic regions should consider the vaccination to prevent the disease. In addition, protective measures should always be taken against mosquito bites, especially during the day when mosquitoes are active, and for those groups, such as pregnant women and immunocompromised persons, for whom vaccination should be carefully considered (43).

## Epidemiological situation at different spatial scales:
past and current trends

The epidemiology of CHIK in Europe is characterized by relatively small outbreaks involving local transmission following the introduction of an imported index case. The first European outbreak wa436s reported in Northeastern Italy from June to September 2007. Four other reported outbreaks involving local transmission of CHIK by *Ae. albopictus* have been reported in Europe. Other outbreaks occurred in Italy in 2017 and three in France in 2010, 2014 and 2017, involving from two indigenous cases in France in 2010, up to 436 cases in Italy in 2017. From 2018 to 2020, 770 cases were notified to the ECDC, and 89% were in people who were exposed abroad. The number of cases notified does not present a clear trend, with great variability across countries and years (Figure 2). Considering the period from 2012 to 2020, the trend of CHIK in Europe was characterized by great variability in the number of cases and countries involved, with the highest peak in 2014, due to the high number of cases (788) notified in France, 98% of them travel-associated. United Kingdom, Spain and Germany are the other countries with high number of
cases, in the considered period, mostly travel-associated. Most of the cases reported in 2017 were from Italy, where an autochthonous outbreak occurred (Figure 3). The spatiotemporal dynamics of CHIK outbreaks remain unpredictable in Europe, with most of the cases imported from endemic areas by travellers, and some outbreaks in areas with high vector density.

<figure><img src="image_17.png" /><figcaption>**Figure 2.** Number of reported cases of CHIKV infection in countries of the European Union and European Economic Area, 2018–2020 (85% travel-associated) (data from http://atlas.ecdc.europa.eu/public/index.aspx)</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>**Figure 3.** Number of reported cases of CHIK in countries of the European Union and European Economic Area, 2012-2021 (85% travel-associated) (data from http://atlas.ecdc.europa.eu/public/index.aspx)</figcaption></figure>
## Sociological and demographical dimension affecting susceptibility and exposure, including gender

The occurrence of CHIKV infections, at EU level, arise for several factors. In temperate climate countries, the risk of infection increases in people, due to social and behavioural factors. Globalisation of travel through highly viraemic tourists travelling from endemic countries to non-endemic areas, in combination to risky behaviours that favour exposure to *Ae. albopictus* bites, such as staying outdoors during daytime and scarce adoption of protective behaviours (i.e., use of insect repellents) increase the risk of infection. The highest rates of infection occurred in the oldest, particularly in males (44, 45). Table 4 shows the most important human covariates related to the CHIKV infections, and Figure 4 shows the notification rate of CHIKV infection by gender and age group.

<table><caption>**Table 4.** List of CHIK human covariates reported in the selected papers in decreasing order of importance according to the number of references</caption><thead><tr><th>Human drivers</th><th>n. of papers#(n. 14)</th><th>% of impact*(n. 44)</th></tr></thead><tbody><tr><td><strong>Infection</strong> (cumulative attack rate; average number of symptomatic day; n. of notified cases; CHIKV/antibodies detection)</td><td>27</td><td>61.36%</td></tr><tr><td><strong>Gender</strong> (rates/gender; risk factor/gender; n. of cases/gender)</td><td>8</td><td>18.2%</td></tr><tr><td><strong>Age</strong> (rates/age)</td><td>7</td><td>15.9%</td></tr><tr><td><strong>Economic condition</strong></td><td>1</td><td>2.27%</td></tr><tr><td><strong>Human behaviour</strong> (use of repellents, work or recreational activity)</td><td>1</td><td>2.27%</td></tr></tbody></table>

# number of papers extracted, the number of references per covariate is higher because more covariates may have been extracted from a document; *% calculated on the total number of references

<figure><img src="image_19.png" /><figcaption>**Figure 4.** CHIKV notification rate by gender and age group in the European Union and European Economic Area, 2012-2021 (85% travel-associated) (data from The European Surveillance System (TESSy-ECDC)</figcaption></figure>
# Diagnostic procedures and notification systems used at local, national and European scale

## Diagnosis

Individuals infected by arboviruses can present a wide range of similar clinical manifestations, such as rash, myalgia, exanthema, arthralgia, joint pain, headache, lymph node hypertrophy, neurological impairment and fever especially when Zika Virus (ZIKV) and Dengue Virus (DENV) are co-circulating in the same geographical region (46, 47).

In this context, variations in the clinical presentation of cases can give hints as to the viral aetiology; for instance, the salient and prolonged polyarthralgia, often accompanied by rash, is typically more indicative of CHIKV, while haemorrhagic manifestations and myalgia are more commonly observed in DENV infections (48).

Since the variety and intensity of symptoms associated to CHIKV, DENV and ZIKV infections are so similar, laboratory analysis is necessary to confirm the respective viral aetiology as laboratory tests for specific diagnosis of CHIKV infection are based on virus isolation, viral RNA detection and serology (49). Molecular methods of CHIKV diagnosis – such as Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), Reverse Transcriptase Loop Mediated Isothermal Amplification (RT-LAMP), quantitative RT-PCR (qRT-PCR) – have gained increasing importance. They are more sensitive and faster than viral isolation, and permit RNA detection from all CHIKV lineages with high specificity. Usually, serum samples collected up to seven days of symptom-onset are suitable for CHIKV detection by molecular diagnostic platforms (50, 51).

In later phases of infection, CHIKV detection is usually based on serological methods, such as ELISA (Enzyme-Linked Immunosorbent Assay) techniques which are useful to distinguish between acute or convalescent infections via detection of anti-CHIKV IgM or IgG antibodies. IgM can be detected from two/four days up to three months after the onset of illness, while IgG can be detected for several years (52).

## Infrastructure capacity to identify pathogens for each Member State

CHIK is among the communicable diseases that according to the Commission Implementing Decision (EU) 2018/945 are covered by epidemiological surveillance. It means that EU Member States are required to establish national capacity of detection and reporting of human cases. The decision provides a case definition and laboratory criteria:

1. Probable case

a. Detection of CHIK specific IgM antibodies in a single serum sample.

2. Confirmed case at least one of the following four:

a. Isolation of CHIKV from a clinical specimen;

b. Detection of CHIKV viral nucleic acid from a clinical specimen;

c. Detection of CHIK specific IgM antibodies in a single serum sample and confirmation and by neutralization;

d. Seroconversion or four-fold antibody titre increase of CHIK specific antibodies in paired serum samples.
Diagnosis is routinely made by clinical microbiology laboratories, and there is no European-
wide reference laboratory network or national laboratories in most EU countries.

Estimated influence of environmental change
on the disease future trends

The current risk of CHIKV transmission in Europe is not primarily restricted by temperature, which allows extrinsic incubation of the virus, but rather by the distribution and density of competent vectors.

Some risk scenarios for Europe were developed, with highly suitable areas more widespread than previously assumed. Coastal areas of the Mediterranean Sea, in the western part of the Iberian Peninsula, and in Atlantic coastal areas of France are those at highest risk, but under a worst-case scenario, even large areas of western Germany and the Benelux states are considered potential areas of transmission (53). However, some parts of the regions of highest current suitability, eg. northern Italy near the Adriatic coast, are projected to experience a decline in suitability due to increased probabilities of summer droughts, which will reduce the habitat suitability for the vectors (54).

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# CRIMEAN-CONGO HAEMORRHAGIC FEVER

Soushieta Jagadesh (a), Claudia Cataldo (b), Francesca Dagostin (c), Ferran Jori Massanas (d, e), Annapaola Rizzoli (c), Luciano Toma (f), William Wint (g), Luca Busani (b)

(a) *International Society of Infectious Diseases, Boston (MA)*

(b) *Centro di Riferimento per la Medicina di Genere, Istituto Superiore di Sanità, Rome*

(c) *Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento*

(d) *Cirad, UMR Astre, Montpellier*

(e) *University of Montpellier, INRAE, Montpellier Centro Ricerca e Innovazione, Fondazione Edmund Mach, San Michele all'Adige, Trento*

(f) *Dipartimento Malattie Infettive, Istituto Superiore di Sanità, Rome*

(g) *Department of Biology, Environmental Research Group Oxford Ltd, Oxford*

## Biological, ecological and molecular features of the causative agent

### Disease name

Crimean-Congo Haemorrhagic Fever (CCHF).

### Disease agent

#### Common scientific and Latin name

Crimean-Congo hemorrhagic fever virus (CCHFV) is the etiological agent of CCHF, a tick-borne disease transmitted by Ixodid ticks mainly of the genus *Hyalomma* (in particular *H. marginatum*, *H. anatolicum*, *H. rufipes* and *H. asiaticum*) that are both reservoirs and vectors (1, 2). CCHFV was first identified in the Crimean region, Russia, in 1944 and was subsequently shown to be identical to the Congo virus identified in the Congo basin in 1956, giving the virus its current name (1, 3, 4).

### Taxonomy

CCHFV belongs to the genus *Orthonairovirus* of the family *Nairoviridae* within the order *Bunyavirales* (5,6). In addition to CCHFV, the *Nairoviridae* family consists of arthropod-borne viruses causing enzoonotics such as Nairobi sheep disease virus (NSDV), Dugbe virus and Hazara virus (HAZV), with some rare cases in humans reported (7). The *Bunyaviridae* family contains over 350 named isolates classified within five genera, namely, *Hantavirus*, *Nairovirus*, *Orthobunyavirus*, *Phlebovirus*, and *Tospivirus* (8). *Bunyaviridae* family together with members of the *Arenaviridae* and *Orthomyxoviridae* families, are known as segmented negative-strand RNA viruses (sNSVs) attributed to their multiple genome segments (9).

### Disease agent characteristics

CCHFV is an enveloped virus of 80-120 nanometres (nm) in diameter with a single negative-sense RNA (9, 10) with a three-segmented genome, small (S), medium (M), and large (L) RNA segments, which encode a viral nucleocapsid protein (NP), glycoprotein precursor, and
polymerase proteins, respectively (11). The genome size of CCHFV was found to be approximately 19.2 kilobases (kb) in length, consisting of a 1.7-2.1 kb S segment, a 4.4-6.3 kb M segment and a 11-1.4 kb L segment (9, 12). CCHFV NP possesses a racket-shaped overall structure with dimensions of 40 × 50 × 95 Å, and features two major parts: a “head” domain and a “stalk” domain (13). Although the NP and L proteins of CCHFV strains are conserved at approximately 95% or more, the CCHFV GPC is much less conserved, with divergent strains exhibiting less than 75% amino acid conservation (14, 15). The genetic diversity of CCHFV correlates with geography (15, 16). CCHFV is classified into seven clades/genotypes (I to VII): Africa-1 (genotype I), Africa-2 (genotype II), Africa-3, (genotype IIIa) and Africa 4 (genotype IIIb); Asia-1 (genotype IVa) and Asia-2 (genotype IVb); Europe-1 (genotype V), Europe-2 (genotype VI) and Europe-3 (genotype VII) (15) based on the S segment sequences with genotype IV divided further into two subgenotypes.

## Physiochemical properties

CCHFV is classified as a biosafety level 4 (BSL4) pathogen (17). As an enveloped virus, CCHFV can be inactivated by suitable disinfectant solutions including chlorine-based disinfectants like 1% sodium hypochlorite (18, 19), 40% ethanol within 2 min (20), 10% aqueous solution of household bleach, 2% solution of glutaraldehyde (18), phenolic disinfectants (0.5%-3%) such as formalin and paraformaldehyde, and other disinfectants, such as hydrogen peroxide and peracetic acid (21). The virus gets inactivated by dry heat at 56°C for 30 minutes or 60°C for 15 minutes (22). Ultraviolet exposure (1,200 to 3,000 µW/cm²) or low pH (less than 6) also inactivates the virus (1, 21). CCHFV is stable for upto 10 days in blood kept at 40°C (23). The virus is stable under wet conditions for 7 hours at 37 °C, 11 days at 20 °C and 15 days at 4 °C (20) while under dry conditions, it is stable for at least 90 min, but less than 24 hours (24). Infectivity of CCHFV is also destroyed by boiling or autoclaving (25).

## Priority level for European Union

CCHF is classified as a priority disease for the European Union (EU) because of its epidemic potential, its high case fatality ratio, its potential for nosocomial outbreaks, and the difficulties in treatment and prevention. CCHF in humans is a notifiable disease at the EU/EEA (European Economic Area) level (26) and it was included in the WHO R&D Blueprint priorities for research and product development in 2016 due to its epidemic potential and insufficient countermeasures against CCHFV (27, 28). Within the EU, CCHF is currently reported in Bulgaria, Spain, and Greece, but disease activity is documented in neighbouring countries Albania, Georgia, Kosovo, Russia, Ukraine, and Turkey (29). In Bulgaria, CCHF is considered endemic since the 1950's (30, 31). Imported cases have been reported in France (32), Greece (33), and Germany (34). In 2018, Greece reported its first and only autochthonous case of Crimean-Congo haemorrhagic fever (CCHF) (35, 36). Spain reported its first case in 2016 in the province of Ávila, Castile-León (37). A retrospective study observed that a case of CCHF had occurred in the same province in 2013 (38).

## Distribution of the pathogen

The geographic distribution of CCHFV is directly linked with the distribution of *Hyalomma* ticks, having a 50° north latitude limit. The virus has been detected across a wide geographic
range in more than 50 countries of Asia, Europe, and Africa where it is associated either to outbreaks of haemorrhagic fever or only sporadic cases, making CCHF the most geographically widespread viral tick-transmitted haemorrhagic fever (3, 4, 39-41). The geographic distribution of CCHF spans from western China, across Southern Asia to the Middle East, Spain, the Balkans, and most of Africa (14, 42, 43) (Figure 1). CCHFV has been reported in Asia (Iran, Afghanistan, Pakistan, Iraq, United Arab Emirates, Kuwait, Oman, Saudi Arabia, China, Tajikistan, Uzbekistan, Kazakhstan, India) (44), Africa (South Africa, Egypt, Mauritania, Kenya, Sudan, Democratic Republic of Congo, Chad, Niger, Nigeria, Senegal, Uganda, Tanzania) (45), and Europe (Albania, Bulgaria, Turkey, Greece, Georgia, Russia, Kosovo, Spain, North Macedonia) (46). To date, CCHF has not been reported in northern Europe, Australia or in the Americas.

<figure><img src="image_20.png" /><figcaption>Figure 1. Distribution of CCHF in humans in 2020</figcaption></figure>

The endemicity of CCHFV corresponds to the broad distribution of *Hyalomma* ticks, the predominant vector and reservoir of the virus (47-49) and are considered as crucial in maintaining endemic foci (1, 14, 43). The geographic distribution of CCHF is directly linked with the distribution of *Hyalomma* ticks, having a 50° north latitude limit. *Hyalomma marginatum* remains the main vector of CCHFV in Europe, it is found in Albania, Bulgaria, Cyprus, France, Greece, Italy, Kosovo, Moldavia, Portugal, Romania, Russia, Serbia, Spain, Turkey, and the Ukraine, and is well adapted to a wide range of abiotic conditions but it prefers rather arid localities with high summer temperatures (1) (Figure 2). *H. lusitanicum* ticks have been found to play an important role of CCHFV persistence in Spain (50,51) and Portugal (Figure 3).

CCHV has also been isolated from *Dermacentor marginatus* tick collected in wild boars in Spain (95).
<figure><img src="image_21.png" /><figcaption>Figure 2. Current 1-km probability of presence of *H. marginatum* across Europe, produced using random forest and boosted regression trees analyses (source: updated by ERGO group)</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>Figure 3. Current 1-km probability of presence of *H. lusitanicum* across Europe, produced using random forest and boosted regression trees analyses (source: updated by ERGO group)</figcaption></figure>
Ecology and transmission routes

CCHFV is transmitted through an enzootic sylvatic cycle: tick – non-human vertebrate – tick. Hard bodied or ixodid ticks, mostly of the *Hyalomma* genus, are considered biological vectors, as well as reservoirs for CCHFV, as they can maintain the virus for several months or even years (1). CCHFV has been detected or isolated from additional tick species, but studies are needed to show whether they are competent virus vectors or merely coincidental unmaintained tick infection from recent feeding on an infected animal or co-feeding (feeding on an uninfected vertebrate host in proximity with an infected tick) (52). They are also able to transmit CCHFV from one generation to the next (vertical transmission), from one development stage to the other (transovarial transmission), from males to females during copulation (sexual transmission), or from one tick to other ticks feeding closely on a same non-viremic host (cofeeding) (43, 53). After infection, ticks remain infective during their lifespan.

The main vector of CCHFV seems to be *H. anatolicum* in Near East and Middle East, *H. asiaticum* from Central Asia to China, *H. rufipes* in Southern Russia and Africa and *H. marginatum* in the Europe and some parts of Asia and Africa. In Spain, CCHFV was detected in *H. lusitanicum* ticks before the identification of the first human case, and studies have observed that this tick species plays an important role in virus circulation in the country (50, 51).

In CCHF endemic areas, where the climatic and environmental factors are suitable for *H. marginatum* ticks (and their animal hosts), their population is increased in spring and summer, accounting for >30% of tick species in the area. *H. marginatum* needs the presence of vertebrate hosts to complete the blood meals required to molt from one development stage to the next. It is a ditropic tick meaning that it is a two-host species. The larvae and nymph stages feed on the same host species, which are small vertebrates such as lagomorphs, birds, hedgehogs, and rodents, whereas adult stages usually feed on large ungulates such as horses, cattle, sheep, goat, deer or wild boar, and occasionally humans (54).

Non-human vertebrate hosts are not symptomatic, but they can replicate the virus and be a source of infection for both ticks and humans (55) during the viremia phase, that lasts 2-15 days (2). CCHFV has been described vertebrate species and is found to circulate according to the geographical distribution of its tick vectors and different sedentary or migratory vertebrate hosts, which are amplifiers of virus (1). *Hyalomma* ticks feed on a variety of mammals including domestic ruminants such as sheep, goats and cattle, rodents, lagomorphs, and wild herbivores (56). Infection in mammals, although subclinical, produces sufficient viremia levels to enable CCHFV transmission to uninfected ticks (57).

Domestic animal species have been implicated in CCHFV transmission to humans. In endemic regions, sheep have been recognized as CCHFV reservoirs and have been epidemiologically linked to human cases (58-61).

In Uzbekistan, three CCHF cases were described in persons in contact with a sick cow (62). Similarly, the first patient in an epizootic of CCHFV in Mauritania became ill following butchering a goat (63).

Studies have shown that increased CCHFV IgG seropositivity in livestock often parallels reports of CCHF cases in humans with exposure to livestock (e.g., slaughterers, butchers, and farmers), particularly in those who handle blood and organs from infected livestock (31,64-68).

Cattle are noted to be the most sensitive indicator of low-level CCHFV circulation as they tend to be highly infested with *Hyalomma* spp. ticks, the numbers of which can be ten times higher than those found on small ruminants (69) (Table 1).
<table><caption>Table 1. List of European countries that reported seroprevalence for CCHFV in domestic animals</caption><thead><tr><th>Animal</th><th>Country</th><th>Seroprevalence %</th><th>Assay</th></tr></thead><tbody><tr><td rowspan="15">Cattle</td><td>Albania (ten regions) (70)</td><td>4.74</td><td>IgG ELISA</td></tr><tr><td>Albania (Berat) (71)</td><td>4</td><td>IgG ELISA</td></tr><tr><td>Albania (Gjirokastra) (53)</td><td>2.1</td><td>IgG ELISA</td></tr><tr><td>Albania (Kolonje) (71)</td><td>7.4</td><td>IgG ELISA</td></tr><tr><td>Albania (Rreshen) (53)</td><td>2.6</td><td>IgG ELISA</td></tr><tr><td>Armenia (72)</td><td>4.2</td><td>AGDP</td></tr><tr><td>Bulgaria (73)</td><td>33.2</td><td>AGDP</td></tr><tr><td>Bulgaria (Aytos) (74)</td><td>71</td><td>IgG ELISA</td></tr><tr><td>Hungary (75)</td><td>0.9</td><td>AGDP*</td></tr><tr><td>Ireland (76)</td><td>1.9</td><td>RPHI</td></tr><tr><td>Kosovo (77)</td><td>18.4</td><td>IgG ELISA</td></tr><tr><td>Republic of North Macedonia (78)</td><td>14.6</td><td>IgG ELISA</td></tr><tr><td>Russia (Astrakhan Oblast) (79)</td><td>5.1</td><td>AGDP</td></tr><tr><td>Russia (Rostov Oblast) (80)</td><td>23</td><td>AGDP</td></tr><tr><td>Russia (Rostov Oblast) (81)</td><td>2.8</td><td>AGDP</td></tr><tr><td>Russia (Rostov Oblast) (82)</td><td>0.5-17.0</td><td>AGDP</td></tr><tr><td rowspan="5">Goats</td><td>Albania (83)</td><td>20</td><td>IgG ELISA</td></tr><tr><td>Bulgaria (73)</td><td>62.3</td><td>AGDP</td></tr><tr><td>Bulgaria (Aytos) (74)</td><td>60</td><td>IgG ELISA</td></tr><tr><td>Kosovo (77)</td><td>10</td><td>IgG ELISA</td></tr><tr><td>Turkey (marmara) (84)</td><td>66</td><td>IgG ELISA</td></tr><tr><td rowspan="3">Horses</td><td>Bulgaria (73)</td><td>39</td><td>AGDP</td></tr><tr><td>Russia (Astrakhan Oblast) (79)</td><td>3.1</td><td>AGDP</td></tr><tr><td>Russia (Rostov Oblast) (80)</td><td>Pos</td><td>AGDP</td></tr><tr><td rowspan="2">Sheep</td><td>Bulgaria (73)</td><td>32.9</td><td>AGDP</td></tr><tr><td>Bulgaria (Aytos) (74)</td><td>74</td><td>IgG ELISA</td></tr><tr><td rowspan="2">Donkeys</td><td>Bulgaria (73)</td><td>62.3</td><td>AGDP</td></tr><tr><td>Bulgaria (Aytos) (74)</td><td>60</td><td>IgG ELISA</td></tr><tr><td>Camels</td><td>Russia (Astrakhan Oblast) (85)</td><td>1.4</td><td>AGDP</td></tr><tr><td>Misc. small livestock</td><td>Kosovo (excluding sheep) (59)</td><td>14</td><td>IgG ELISA</td></tr></tbody></table>

ELISA Enzyme-Linked Immunosorbent Assay

AGDP Agar gel Diffusion Precipitation

Considerable seroprevalence was consistently reported in wild animals as reported with hares (3-22%), buffalo (10-20%), and rhinoceroses (40-68%) (53). A substantial tick load of up to 40 larval and nymphal *H. marginatum* ticks has been described on hedgehog hosts (*Erinaceus europaeus*, *Hemiechinus auritus*) during the peak season of immature tick activity (1, 86). However, the role of hedgehogs in enzootic maintenance appears to be variable by species. *Hemiechinus auritus* developed viremia during experimental infection (87) and are considered a natural CCHFV reservoir by serving as a source of CCHFV for feeding ticks.
In contrast, in the same study, experimental infection in the European hedgehog (*E. europaeus*) did not produce detectable viremia, suggesting reduced susceptibility to infection or more efficient viral clearance.

Among the EU/EEA countries, seroprevalence studies have demonstrated the presence of antibodies to CCHFV in bats in France (88, 89), and in hares in Bulgaria (73) and Hungary (90) (Table 2). Many bird species are important hosts for *Hyalomma* ticks and can transport them over long distances (91, 93). However, most birds are resistant to infection except for ostriches (94).

<table><caption>Table 2. Wild animals testing positive for antibodies against CCHFV in Europe</caption><thead><tr><td>Class</td><td>Common name</td><td>Species</td><td>Country (ref.)</td><td>Seroprevalence</td></tr></thead><tbody><tr><td>Aves</td><td>Eurasian magpie</td><td>Pica pica</td><td>Russia (95)</td><td>1 animal</td></tr><tr><td>Mammalia</td><td>Red fox</td><td>Vulpes vulpes</td><td>Russia (95)</td><td>-</td></tr><tr><td>Mammalia</td><td>Bats</td><td>Various spp.</td><td>France (88, 89)</td><td>10.5% (2/19)</td></tr><tr><td>Mammalia</td><td>European hare</td><td>Lepes europaeus</td><td>Russia (95)</td><td>20%</td></tr><tr><td>Mammalia</td><td>European hare</td><td>Lepes europaeus</td><td>Hungary (90)</td><td>6% (12/198)</td></tr><tr><td>Mammalia</td><td>Hare</td><td>Lepes spp.</td><td>Bulgaria (73)</td><td>3% (1/33)</td></tr><tr><td>Mammalia</td><td>Wild boar</td><td>Sus scrofa</td><td>Spain (96)</td><td>19.4%</td></tr><tr><td>Mammalia</td><td>Red deer</td><td>Cervus elaphus</td><td>Spain (96 97)</td><td>25.4</td></tr><tr><td>Mammalia</td><td>Roe deer</td><td>Capreolus capreolus</td><td>Spain (98)</td><td>1.2% (1/79)</td></tr><tr><td>Mammalia</td><td>Iberian Ibex</td><td>Capra hispanica</td><td>Spain (98)</td><td>78% (66/84)</td></tr><tr><td>Mammalia</td><td>Mouflon</td><td>Ovis musimon</td><td>Spain (99)</td><td>100% (48/48)</td></tr></tbody></table>

CCHFV is transmitted to humans by bites from infected ticks or by direct contact with blood or tissues of infected ticks, viraemic patients or viraemic livestock. There have been a few reports of infection after consumption of raw meat (100-102). Drinking unpasteurized milk is also mentioned to be a risk factor in CCHFV transmission. CCHFV is usually inactivated in meat due to post-slaughter acidification (3).

Hospital-acquired infections can occur due to direct contact with blood or tissues of viremic patients or improperly sterilized medical devices (103). Human-to-human transmission usually occurs in hospital settings with a high risk of transmission to healthcare workers (HCWs) including doctors, nurses, laboratory staff, research scientists, emergency service staff and cleaning personnel (103). Several nosocomial outbreaks have been attributed to hospitalized patients acting as index cases (104-106).

Retrospective analysis from Turkey has demonstrated that needle stick injuries are the most frequent cause of nosocomial exposures, followed by ‘splash’ exposures to mucous membranes (107). Horizontal transmission of the CCHFV from a mother to her child (107), as well as intrauterine infection (109) have been reported. Transmission of CCHFV during aerosol-generating medical procedures (105) or sexual contact (110) may be possible.
# Drivers of the disease emergence and spread

## Ecological drivers

CCHF, as other vector-borne viral diseases, is influenced by dynamic factors such as climate change, alterations of land use, habitats fragmentation, loss of biodiversity, and introduction of new species that impact the distribution of the vector and hosts (91, 111, 112). Studies have observed increased incidence of CCHF with increasing mean temperature in endemic areas (113-115). The seasonal pattern and abundance of *H. marginatum* was found dependent on temperature (116, 117). High temperatures, especially in the spring and summer, tend to accelerate *H. marginatum* cycle by switching on its interstadial development (118), and increase host questing activity (3, 116, 118). Also, the risk of exposure to ticks for humans is higher in warmer temperature due to increased recreational and outdoor activities (119, 120). Areas regularly experiencing long periods of low rainfall and humidity were associated with increased occurrence of CCHF in Iran (121) and Senegal (122).

In temperate areas, the pattern of seasonality of CCHF cases reflects the period of the year with high tick activity; between spring and early autumn (123-126). Mild winters were followed by CCHF outbreaks in Kosovo in 2001 and in Turkey in 2004 (3).

The incidence of CCHF is higher in those areas characterized by a high proportion of grasslands, scrub, and herbaceous vegetation (savannah-type environment), the environmental niche for *Hyalomma* ticks (113, 127). Studies in Turkey and Greece found non-irrigated agricultural land cover (e.g., pasture and rangeland) to be associated with CCHF incidence (66, 128). Areas with a higher incidence of CCHF were those characterized by a highly fragmented habitat (113, 129, 130), supporting the hypothesis that a fragmented land structure may increase the risk of acquiring CCHF by favouring viral circulation and amplification through frequent at-risk contacts between ticks, humans, livestock and wildlife (129). Deforestation has been hypothesized to increase the risk of re-emergence of CCHF in Central Africa as the local CCHFV persistence is supported by the sylvatic natural cycle (48). Landscape modifications such as disruption of agricultural activities and expansion of the hare population infested with infected ticks, followed by the reintroduction of cattle and sheep, have been associated with the CCHF outbreaks in the former Soviet Union, Bulgaria, Kosovo, and Turkey (3).

The impact of biodiversity loss and its consequent dilution effect on CCHF remains relatively unknown as the disease characterized by a variety of different transmission and tick hosts. However, the rise in the wild boars and deer population densities could facilitate the spread of CCHFV through a parallel increase in tick numbers and dispersion across Europe (131). Migration of animals is strongly impacted by climate warming, and changes in host migratory patterns have important consequences for infectious diseases (132, 133). The CCHFV infection in livestock was found to be a strong positive predictor of CCHF incidence in humans in Iran (58) and Mauritania (134). However, in Bulgaria where vaccination coverage is high amongst at-risk populations (e.g., veterinarians and farm workers), livestock density was not found to be a significant driver of CCHF incidence in humans (113). Livestock transportation outside the safety regulations is also reported in the spread of CCHF (126).

Studies demonstrate a significant positive association between Normalized Difference Vegetation Index (NDVI) and CCHF incidence (113, 135), suggesting that the NDVI may proxy for tick seasonal activity. Socioeconomic and demographic factors influencing CCHF outbreaks include social disruption, conflict, and war (123-126).
# Natural history of disease in humans and animals, including symptoms, morbidity, and mortality

## Brief history of the pathogen and disease

CCHFV introduction to Central and South Asia dated back to the 12th century, which is compatible with historical references describing a disease, which is now believed to have been CCHF, around 1100 AD near Tajikistan in Middle Asia (1). In 20th century, CCHF was described for the first time among Soviet Union military personnel in Crimea during World War II (1944-45) and was named Crimea haemorrhagic fever. The virus was later isolated from blood and tissues of patients using intracerebral inoculation of suckling mice in 1967 (136). The virus responsible for Crimea haemorrhagic fever was found identical to Congo virus that caused febrile illness in Belgian Congo. Later, the two names were combined to new nomenclature of the CCHFV in 1969 (136). Following its description in 1967, cases were reported from former USSR (Crimea, Astrakhan, Rostov), Uzbekistan, Kazakhstan, Tajikistan, and Bulgaria in Eurasia (1, 136) and from Democratic Republic of the Congo, Uganda, and Mauritania in Africa (40, 138). Sporadic cases were reported in Iraq (139), the United Arab Emirates (UAE) (140), and Saudi Arabia (141) in the late 20th century. In Western Europe, the presence of the virus had only been detected indirectly by seroprevalence assays in the serum of two people from Southern Portugal in the early 1980s (142). At the start of 21st century, emergence of CCHF was reported from Pakistan (143), Iran (144), Bulgaria (123), Turkey (41, 145, 146), and India (147). In western Europe, the Greece reported its first autochthonous case in 2008 (35, 147) CCHFV was detected in ticks from deer captured in western Spain in 2010 (50), sheep from Portugal (149), and ticks from birds migrating from Morocco in 2013 (150) prior to detection of their first case in 2016 (37).

Phylogenetic studies have shown that there have been only two virus introductions to Europe (151). The first genotypic group was introduced to Europe via the Volga Delta region only a few hundred years ago and spread to the Balkans. The second introduction was via Turkey around a century ago. A third introduction occurred more recently when the African III genotype was introduced by migratory birds from West Africa in Spain (150).

## Disease in humans

In humans, CCHF characterized by fever and haemorrhage, often with nonspecific signs and symptoms with case fatality rates (CFR) ranging from 5-30% (3,14). However, seroprevalence studies show that most CCHF cases (>80%) are asymptomatic or mild (60, 152). The disease presents in four distinct phases: incubation, pre-haemorrhagic, haemorrhagic and convalescence (1, 40, 41, 153). The incubation period is usually less than a week (range 1-9 days) and is dependent on the route of exposure and virus dose. The incubation period is the shortest following a tick bite/needlestick (usually 1-3 days) and slightly longer following exposure to blood, tissue and secretions of infected livestock and humans (5-6 days). The pre-haemorrhagic stage lasts about 2-4 days on average (range 1-7 days) and begins abruptly with nonspecific prodromal symptoms including fever (39-41°C), headache, myalgia, dizziness, neck pain and stiffness, backache, headache, sore eyes and photophobia (153). This may be accompanied by sore throat, abdominal pain, nausea, vomiting and non-bloody diarrhoea (1, 153). Hyperaemia/ cutaneous flushing of the face, neck and chest, congested sclera and conjunctivitis, and jaundice may also be noticed (1). In severe cases, neurological changes in mood and sensory perception have been reported. Somnolence may replace agitation (154, 155). In most patients, the pre-haemorrhagic
phase progresses to haemorrhagic phase. On examination, hypotension, relative bradycardia, tachypnea, and Hepatomegaly and splenomegaly may also be present (41, 145, 156, 157).

The haemorrhagic stage is usually short (approximately 2-3 days) but can be prolonged up to two weeks (153). Haemorrhagic manifestations range from petechiae to extended ecchymoses on mucous membranes and skin (1). These cutaneous signs are particularly pronounced with CCHF compared with other viral haemorrhagic fevers and have found to have a correlation between morbilliform eruptions, platelet count and favourable prognosis (158). Epistaxis, melena, haematemesis, haematuria and haemoptysis are common as is bleeding from injection sites (153, hyju154). Case reports of haemorrhage in other sites such as the vagina (159), uterus (160) and brain (161) have been reported. The haemorrhagic stage is pronounced in severe cases, with rapid progression to disseminated intravascular coagulation (DIC), overt bleeding, kidney, liver or pulmonary failure, and shock (104, 152 153, 162). Acute Respiratory Distress Syndrome (ARDS) and diffuse alveolar haemorrhage have also been reported during haemorrhagic manifestations (163, 164). Death usually occurs in the second week of illness (153). At this stage, laboratory tests demonstrate thrombocytopenia, leukopenia and elevated levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase and creatine phosphokinase (153, 155, 165-167) along with elevated levels of inflammatory cytokines (168, 169). Studies show that coagulation is affected, with prolonged prothrombin and activated partial thromboplastin times accompanied by a decrease in fibrinogen levels and an increase in the levels of fibrinogen degradation products (153, 155, 165, 167).

In survivors, convalescence generally begins around 9-10 days post-onset of illness (range 9-20 days) and is associated with a return to normal for laboratory parameters (40, 145, 53). This stage can be prolonged and may be associated with hypotension, tachycardia or bradycardia, polyneuritis, sweating, headache, dizziness, nausea, poor appetite, breathing difficulties, xerostomia, vision and hearing deficiencies, hair loss and memory loss among others are rarely permanent, but may persist for a year or more (1). However, there is insufficient evidence to describe the long-term complications, sequelae and disability associated with CCHF (170). Post-traumatic stress disorder and mild hearing loss have been reported (171, 172). The differential diagnosis of CCHF is broad and is dependent (Table 3) on patient's geographic origin (173).

<table><caption>Table 3. Differential diagnosis of CCHF</caption><thead><tr><th>Criteria</th><th>Differential diagnosis of CCHF</th></tr></thead><tbody><tr><td><strong>Geographic origin</strong></td><td></td></tr><tr><td>Middle East</td><td>Alkhurma haemorrhagic fever and Rift Valley fever</td></tr><tr><td>Russia</td><td>Omsk haemorrhagic fever</td></tr><tr><td>India</td><td>Kyasanur forest disease</td></tr><tr><td>Europe and Asia</td><td>Hantaviral diseases</td></tr><tr><td>Africa</td><td>Lassa virus, Ebola virus, Marburg virus, Rift Valley fever and yellow fever</td></tr><tr><td>Asia and central Africa</td><td>Dengue</td></tr><tr><td>Tropical &amp; subtropical</td><td>Malaria</td></tr><tr><td><strong>Transmission</strong></td><td></td></tr><tr><td>Vector: Tick bite</td><td>Rickettsia spp., Ehrlichia spp., Borrelia, Anaplasma and Babesia</td></tr><tr><td><strong>Clinical symptoms</strong></td><td></td></tr><tr><td>Symptomatology</td><td>Tularaemia, Q fever, viral hepatitis, influenza virus infection, meningococcal meningitis, leptospirosis, typhoid fever, sepsis due to staphylococci or Gram-negative bacilli, toxic shock syndrome, salmonellosis, shigellosis, psittacosis, trypanosomiasis, septic infection due to Yersinia pestis, rubella and measles</td></tr></tbody></table>
## Disease in animals

Most non-human mammals acquire sub-clinical infections and are asymptomatic during and following the period of viremia (55). Most birds are resistant to infection except for ostriches (94).

# Availability of preventive, therapeutic and control measures, including licensed or pipeline vaccines

## Therapy in humans

Symptomatic treatment of CCHF during the pre-haemorrhagic phase includes antipyretics for fever, proton pump inhibitors to prevent gastrointestinal bleeding (3, 174, 175) and in women, progesterone to avoid menorrhagia (174). Intramuscular injections and Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) to be avoided due to the repercussions for clotting (3). Suitable electrolyte replacement must be ensured, and hypotonic solutions are to be avoided (175).

In the haemorrhagic phase, CCHF requires close laboratory monitoring: complete blood count, alanine transaminase (ALT), aspartame aminotransferase (AST), total bilirubin, creatinine, prothrombin time, activated partial thromboplastin time (aPTT), and lactate dehydrogenase. If Disseminated Intravascular Coagulation (DIC) is suspected, d-dimer and arterial blood gas values should be obtained (174, 175).

The efficacy of glucocorticoids for the treatment of CCHF is inconclusive (175) as studies evaluating their usefulness are limited and consist of small case series in both adults and paediatric patients (176). These studies found that administration of high-dose methylprednisolone (20-30 mg/day) seems to promote early haematological recovery, reverse haemorrhagic lesions and decrease the need for transfusion of blood products. However, these results have been inconclusive due to patients simultaneously receiving ribavirin (176, 177). Transfusion with blood products such as platelet concentrates and fresh plasma in the haemorrhagic phase, is effective in cases of complications massive haemorrhage, liver failure, thrombocytopenic thrombotic purpura, dilutional coagulopathy, DIC, INR 1.5 times above normal limits and decreased aPTT (174, 175). There is currently limited evidence regarding treatment with plasma or antibodies from survivors (178, 179) and monoclonal antibodies against viral proteins (180,181).

To date, there are no antiviral drugs with proven efficacy against CCHF virus (182, 183). One of the main problems for research on active drugs against CCHFV is the lack of animal model as CCHFV is not pathogenic in animals. Ribavirin is the antiviral drug with the most extensive experience of use in CCHF, with controversial data (153, 154-156, 183, 184-189). It has demonstrated conflicting efficacy against CCHF virus (190-193). Data on its efficacy is poor in humans, based on observational studies (174, 177, 191), a single open-label clinical trial (194) and two meta-analyses (189, 195). The meta-analyses report that the data supporting the efficacy of ribavirin against CCHFV are poor owing to confounding factors in reported data sets and any benefit probably requires early treatment during the phase of high viremia (196). Favipiravir showed significant protective effects in lethally infected mice (192, 193, 197), preventing death and significantly reducing viral loads in key target tissues of CCHFV, even when initiated days after infection, and in mice with advanced disease. This data suggests that favipiravir may be effective in patients presenting to health-care systems with advanced CCHF. However, further pre-clinical and clinical studies are needed in humans. Molnupiravir, recently used to treat SARS-CoV-2 infection in humans, exhibits efficacy against CCHFV *in vitro* with similar inhibitory
concentrations as favipiravir (197). However, molnupiravir failed to protect against CCHFV infection in lethally infected mice even when treatment was started before infection (197).

## Licensed or pipelined vaccines

To date, multiple vaccine platforms have been evaluated in animal models for CCHFV such as inactivated virus preparations (198), subunit vaccines (199), VLP vaccines (200, 201), recombinant live-attenuated viruses (201-203), replication-deficient viral-vectored vaccines (204) and nucleic acid-based vaccines (205-208). However, clinical trials have not identified any vaccine with proven efficacy (182, 209). Clinical trials are difficult to conduct as outbreaks are sporadic with irregular numbers of cases (178). A vaccine derived from inactivated CCHFV, propagated in mouse brain, is used in Bulgaria (210).

## Other prevention measures

Presently there are no vaccines available for animal protection. CCHF is on the diseases list of WOAH (World Organization for Animal Health). Imports into the EU of live animals are prohibited from endemic areas if the animal tests positive for CCHFV (Commission Regulation (EU) 206/2010).

To reduce the risk of introduction and spread of the diseases from infected countries to non-infected ones there are several measures provided by official regulations, like control of livestock movements from endemic countries (transborder transmission) (211, 212). However, as the infection in animals is asymptomatic, it is difficult to detect and control transmission of CCHFV from animal hosts.

The CCHFV tick vectors are relatively easy to control. Acaricides are useful for tick control when applied prior to animal slaughter, and a 14-day period of quarantine prior to slaughter has also been used (94, 212).

Measures to prevent the infection in humans are based on protection from tick bites. Depending on the geographical location and species, ticks are generally active between April and September in the North Hemisphere. Except during the egg stage, all other biological stages of ticks feed on blood from humans. At risk populations such as agricultural workers and others working with animals including those who live in rural endemic areas should consider basic protection measures (appropriate clothes) and use of tick repellents (4). Minimizing the risk of tick-borne disease transmission with mechanical methods can be performed with tools readily available in most regions (213, 214) such as with tweezers (82.5% success rate) was found to be superior to both lassoing (47.5% success rate) and card detachment (7.5% success rate) (214).

Occupations at high risk for CCHF include veterinarians, abattoir workers, and farmers. For risk reduction in veterinarians and abattoir workers, the use of standard infection control practices when handling potentially infectious blood or ticks was found effective (154). Creating public awareness, targeted at high-risk groups in endemic regions, is useful in reducing the exposure to the virus and controlling the spread of the disease (211, 212).

## Disease specific recommendations

### Reducing the risk of human-to-human transmission

To prevent nosocomial transmission of CCHFV, particularly among health-care workers, early detection and diagnosis of CCHF cases and adequate provision of PPE is essential together with
proper disposal of used instruments and equipment (syringes, needles) (215). However, poor compliance to the recommended PPE for CCHF has previously been reported (216, 217).

At the community level, prompt epidemiological investigation and contact tracing, and safe burial practices must be implemented for CCHF cases. (148, 218).

## Epidemiological situation at different spatial scales: past and current trends

Since 2000, the incidence and geographic range of CCHF cases have markedly increased (44, 194). Turkey has reported approximately 900 new CCHF cases annually, with a total of 9,787 cases reported from 2002 to 2015 (107). CCHF is endemic in the Balkan region, in Kosovo, 228 cases were reported from 1995 to 2013 (77).

Among the EU/EEA countries, the first documented outbreak was reported in Bulgaria occurred during the agricultural collectivization in 1953 (1). After the introduction of a vaccination programme of high-risk groups of the population in 1974 (46), a drastic reduction of cases was observed from 1105 in 1953-1974 to 279 in 1975-1996 (218), to 196 in 1997-2008 (220).

Although the overall number of cases decreased over time, a major outbreak was observed in 2008, with a cluster of cases in the southwest part of Bulgaria, an area historically considered at low risk for CCHF outbreaks (219). In Bulgaria, over 1,500 cases have been reported since 1952 (221). Imported cases have been reported from France in 2004 (32), Germany in 2009 (34), and Greece in 2018 (33). Since its first outbreak in 2016, Spain has reported ten cases of CCHF (Table 4).

<table><caption>Table 4. Cases reported in EU/EEA countries since 2013.</caption><thead><tr><th>Year</th><th>Country</th><th>No. of cases</th><th>Place of exposure</th><th>Transmission</th></tr></thead><tbody><tr><td rowspan="2">2013</td><td>Bulgaria (221)</td><td>8</td><td>Shumen, Yambol, Haskovo, Kardjali and Blagoevgrad regions</td><td>Community</td></tr><tr><td>Spain (37)</td><td>1</td><td>Ávila province, Castile-León</td><td>Community</td></tr><tr><td>2014</td><td>Bulgaria (222)</td><td>8</td><td>Haskovo, Kardjali, Blagoevgrad, Plovdiv and Burgas regions</td><td>Community</td></tr><tr><td rowspan="2">2015</td><td>Bulgaria (223)</td><td>4</td><td>Blagoevgrad, Haskovo and Yambol regions</td><td>Community</td></tr><tr><td>Bulgaria (224)</td><td>4</td><td>Blagoevgrad, Kardjali and Yambol regions</td><td>Community</td></tr><tr><td rowspan="2">2016</td><td rowspan="2">Spain (37)</td><td rowspan="2">2</td><td>Ávila province, Castile-León</td><td>Community</td></tr><tr><td>Madrid province</td><td>Nosocomial</td></tr><tr><td rowspan="3">2017</td><td>Bulgaria (225)</td><td>2</td><td>Kardjali and Haskovo region</td><td>Community</td></tr><tr><td>Bulgaria (226)</td><td>6</td><td>Kardjali and Plovdiv regions</td><td>Community</td></tr><tr><td>Spain (227)</td><td>2</td><td>Badajoz and Salamanca provinces</td><td>Community</td></tr><tr><td>2018</td><td>Bulgaria (228)</td><td>2</td><td>Kardjali region</td><td>Community</td></tr><tr><td rowspan="2">2019</td><td>Bulgaria (229)</td><td>1</td><td>Burgas region</td><td>Community</td></tr><tr><td>Spain (230)</td><td>3</td><td>Salamanca province, Castile-León</td><td>Community</td></tr><tr><td>2020</td><td>Spain (231)</td><td>2</td><td>Salamanca and León provinces</td><td>Community</td></tr><tr><td>2021</td><td>Spain (232)</td><td>2</td><td>León province, Castile-León</td><td>Community</td></tr><tr><td>2022</td><td>Spain (232)</td><td>2</td><td>León province, Castile-León</td><td>Community</td></tr></tbody></table>
## Sociological and demographical dimension affecting susceptibility and exposure, including gender

Human-animal interactions differ across cultures and some interactions may result in social and cultural practices such as the movement of potentially infected animals increasing the risk of CCHFV exposure and linked to CCHF outbreaks (212, 234, 235). The practice of livestock sacrifice plays a major role in festivals like the Hajj and Eid-al-Adha resulting in the contact of large numbers of people with potentially infectious animal blood and body fluids. During Eid-al-Adha, nearly eight million animals are sacrificed each year in Pakistan alone (235) and two million small ruminants, and 750,000 cattle are slaughtered in Turkey, accounting for 25% of all annual slaughtering in that country (235).

Behaviour patterns providing exposure to multifactorial risk factors were gender-based. Men were primarily associated with herding and farming activities, including sleeping outside during seasonal migrations and were more at risk for exposure to CCHFV (236). Seroprevalence studies from Turkey demonstrated anti-CCHFV IgG seropositivity higher in male populations (237, 238). Also, in Spain most of the cases were males, while in Greece, females were more at risk (49) (239). Several studies have shown that increased age was an important risk factor for CCHFV transmission (44, 49, 237, 240-242) and poor prognosis (243). Rural populations are more at risk in terms of exposure to ticks and CCHF.

People living in settlements in rural areas, employed in agricultural activities or in contact with animals (farmers, veterinarians, hunters) are at higher risk of exposure to ticks and CCHF (238, 241, 244-246).

Social disruption, conflict, and war have been the major factors influencing a considerable number of outbreaks in the community and the nosocomial setting (123-126) reported from 2000 to 2008 in South-Eastern Europe (247); in Bulgaria in 2002, 2003 (123), and 2008 (248) and in Albania and Kosovo in 2001.

## Diagnostic procedures and notification systems used at local, national and European scale

### Diagnosis

Early and accurate diagnosis of CCHF is critical for the patient prognosis, early treatment with ribavirin (196), and prevention of nosocomial infection (215). Direct diagnosis by detection of CCHFV RNA using molecular methods or detection of CCHFV antigen and isolation of the virus (BSL 4 laboratory needed) is used in the viremic phase i.e., first week of illness up to day 16 (249). Molecular assays offer a rapid, sensitive and specific diagnosis of CCHF (249). However, the efficacy of molecular methods is affected by high genetic variability of CCHFV strains (250). To mitigate this, it is recommended that nucleic acid amplification tests (NAAT) be used in combination with serological assays, which are less impacted by minor variations (251), for highest detection sensitivity (252-254).

Indirect diagnosis by detection of CCHFV-specific IgM and IgG antibodies from the fifth day of clinical symptoms is accomplished using serological methods such as ELISA (Enzyme-Linked Immunosorbent Assay) or indirect immunofluorescence. These assays target the CCHFV N protein, which induces nearly, strong and long-lasting immune response in humans (194). CCHF infection is confirmed by detection of IgM antibodies or four-fold increase in IgG antibody titres
in serial serum samples (154, 255, 256). Antibody response is often absent or delayed in severe
cases. To date, there are no rapid detection tests for CCHF in development.

CCHF in humans is a notifiable disease at the EU/EEA level (26). All EU/EEA countries have passive surveillance in humans implemented (257). Country specific CCHFV surveillance systems are categorized into five levels based on the incidence of cases, potential for disease transmission to humans, and presence of surveillance systems (221). Level 1 countries are those in which human CCHF cases are reported annually and the virus is endemic (Bulgaria among the EU/EEA countries); Level 2 countries have sporadic autochthonous human cases (Spain and Greece); Level 3 countries have no documented human cases but ecologic data (Portugal and Hungary) with possible human infections; Level 4 countries have the exclusively presence of *Hyalomma* ticks, suggesting the need of seroprevalence surveys (Italy, France, Germany, Austria) and Level 5 countries are ones for which no information is available. Some countries, have in place early detection of exotic ticks and pathogens, some, like in Italy, are based on migratory birds' ticks testing, while others, like in the Netherlands, rely on citizen's reporting of exotic ticks' bites in humans and animals (93, 258).

Infrastructure capacity to identify pathogens
for each Member State

Virus isolation is rarely used for CCHF diagnosis as BSL4 laboratories are required and none
of the European BSL4 laboratories are situated in CCHF endemic areas. Currently, the routine
laboratory diagnosis of CCHF is based on the combination of the detection of the viral genome
and CCHFV specific IgM and IgG as per the international recommendations (4, 253).

There is no official, agreed-upon case definition for CCHF in the EU (148), 22/27 EU/EEA countries use the generic case definition for viral haemorrhagic fevers defined according to the Commission Implementing Decision (EU) 2018/945 of 22 June 2018 for CCHF surveillance.

Bulgaria, Greece, Germany, and Spain have their own case definitions for CCHF which considers as suspect case any patient high fever and one of the following symptoms: severe headache, myalgias, nausea, vomiting, and/or diarrhoea and history of tick bite or history of contact with tissues, blood, or other biological fluids from a possibly infected animal (e.g., abattoir workers, livestock owners, veterinarians) or healthcare workers with a history of exposure to a suspect, probable, or laboratory-confirmed CCHF case, within 14 days prior to the onset of symptoms.

A probable CCHF case is a suspected with thrombocytopenia AND haemorrhagic manifestation. A confirmed CCHF case is laboratory-confirmed by ELISA or ImmunoFluorescence Assay (IFA) for specific antibodies, by RT-PCR for CCHF virus genome or virus isolation (254).

The EU definition of viral haemorrhagic fevers (VHF) is based on clinical criteria (fever or haemorrhagic manifestations), laboratory criteria (virus isolation or detection of specific virus nucleic acid in a clinical specimen and genotyping), and epidemiological criteria (travel or exposure to a case of VHF within the last 21 days).

The case classification includes probable case (clinical criteria with an epidemiological link)
and confirmed case (clinical and the laboratory criteria) (2018/945/EU).
# Estimated influence of environmental change on the disease future trends

Environmental factors and human behaviours are among the most important drivers influencing the lifecycle of *Hyalomma* ticks (220). According to ecological models ran by the World Health Organization Eastern Mediterranean Region, increasing temperature and decreasing rainfall will expand the suitable habitat for *Hyalomma* ticks and will subsequently increase the risk of CCHF (44).Studies predict that trend toward warmer temperatures in central and northern Europe might permit CCHFV to expand outside its current geographic range, through the introduction of infected *Hyalomma* or other reservoir ticks by migratory birds or the international livestock trade (36, 118, 259, 260) A recent study forecasted that the number of countries that have yet to record CCHF have areas that are environmentally suitable for the disease, especially those with Mediterranean coastlines (France, Italy, Southern Balkans) (261). For this reason, future seroprevalence studies in animals should focus on CCHFV presence in Spain, Southern France, Italy, Hungary, and Slovakia (221). Different domestic and wild animal species can be infected by CCHFV, and seroprevalence studies conducted in areas without circulation of the disease have demonstrated seropositivity in cattle, sheep and goats, potentially due to undetected virus circulation between the abundant population of tick vectors and the domestic animals (53, 262).

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