Euroroundups European survey on laboratory preparedness , response and diagnostic capacity for Crimean-Congo haemorrhagic fever , 2012

M D Fernandez-García (mdfernandez@isciii.es)1,2,3, A Negredo1, A Papa4, O Donoso-Mantke5,6, M Niedrig5, H Zeller3, A Tenorio1, L Franco1, the ENIVD members7 1. National Microbiology Centre, Instituto de Salud Carlos III, Madrid, Spain 2. European Public Health Microbiology Training Programme (EUPHEM) 3. European Centre for Disease Prevention and Control (ECDC), Stockholm, Sweden 4. Department of Microbiology, National Reference Centre for Arboviruses, Medical School, Aristotle University of Thessaloniki, Thessaloniki, Greece 5. Zentrum für Biologische Gefahren und Spezielle Pathogene (ZBS-1), Robert Koch Institute, Berlin, Germany 6. Present address: Gesellschaft fuer Biotechnologische Diagnostik mbH, Berlin, Germany 7. The ENIVD members are listed at the end of the article


Introduction
Crimean-Congo haemorrhagic fever (CCHF) is a zoonotic viral disease caused by the tick-borne CCHF virus (CCHFV), which is classified into the genus Nairovirus within the Bunyaviridae family.In humans, the disease is highly pathogenic and life-threatening as it can cause severe illness with prominent haemorrhages reaching case fatality rates of up to 50%.In nature, CCHFV usually circulates between asymptomatic animals and ticks in an enzootic cycle.Humans may become infected through the bite of a tick, mainly of the Hyalomma genus, through direct contact with blood or tissues from viraemic livestock or through direct contact with the blood or secretions of a viraemic patient [1].Thus, risk groups include individuals with outdoor activities, mainly those who have occupational contact with animals, as well as healthcare workers in hospital settings (nosocomial hazard).Because of the potential for epidemics and nosocomial outbreaks, high fatality ratio, limitations for treatment and the lack of safe vaccine, CCHF is a disease listed for immediate notification to public health authorities as it constitutes a major threat to public health.Therefore, CCHFV is considered a high-risk pathogenic organism and classified as a biosafety level (BSL) 4 containment agent.
The disease is endemic in wide areas of Africa, the Middle East, central and south-western Asia and the south-eastern European region.More particularly, some Balkan countries (e.g.Albania, Bulgaria, Greece and Kosovo under UN Security Council Resolution 1244) are endemic zones for CCHF [2].During the last decade, CCHF re-emerged in Albania, Greece, Kosovo under UN Security Council Resolution 1244 and countries bordering the Black sea: Georgia, south-western Russia Turkey, and Ukraine.In Greece, the detection of the non-pathogenic strain AP92 in ticks in 1975 was followed by the notification of the first human CCHF case in June 2008 [3].However, the vast majority of CCHF cases have been recorded in Turkey (since 2002) and the south-western regions of Russia (since 1999), with expanding outbreaks and increasing numbers of associated fatalities [2].In northern and south-western Europe, no human cases have been reported except for imported ones in France [4], Germany [5] and the United Kingdom [6].Limited serological evidence in humans has been reported in parts of Hungary and Portugal [7,8].
In Europe, the tick vector most commonly associated with CCHFV is Hyalomma marginatum, which is present in southern Europe and has sporadically been detected in southern Germany, the Netherlands and the United Kingdom following expansion of its geographical range associated with movement of migrant breeding birds [9][10][11][12].The spreading of the vector represents a risk factor for introduction of the virus from endemic to unaffected areas of Europe, increasing the occurrence of CCHF [13].However, virological evidence has never been addressed in western Europe until 2010, when a study conducted in Spain detected for the first time CCHFV in populations of H. lusitanicum collected from indigenous deer [14].Moreover, the recent discovery of antibodies against CCHFV in livestock in Romania, with prevalence values similar to those observed in other regions where the disease is endemic, suggests an extension of the circulation zone of CCHFV in Europe [15].
In 2008, after the first case in Greece was detected, the European Centre for Disease Prevention and Control (ECDC) organised an expert consultation on CCHF to identify preparedness interventions in Europe [13].In 2011, under the initiative of the European Network for Diagnostics of 'Imported' Viral Diseases (ENIVD; www.enivd.org),a multicenter study of CCHF diagnostic tests and an external quality assessment

Participation
Sixty-eight laboratories from 37 countries (28 EU Members States and nine countries outside the EU), were contacted for this survey.Thirty-one laboratories from 28 countries returned their answer, except Albania, Bosnia and Herzegovina, Cyprus, Denmark,  qRT-PCR 3 [20] qRT-PCR 2 [19] qRT-PCR 1 [18] Own Finland, Hungary, Ireland, Luxembourg and Poland, corresponding to a participation of 76% of the countries and 45% of the laboratories.Of all laboratories that participated in the survey, 21 acted as a NRL for VHF and five act as a WHOCC.

Preparedness and response
All respondent laboratories declared that CCHF was a notifiable disease in their countries and that they followed the generic case definition for VHFs, while six countries (Bulgaria, Greece, Germany, Turkey, Russia and Spain) had their own case definition for CCHF (Table 2).
Most laboratories (25/31) stated that they had trained staff authorised to handle CCHF samples and that there was trained staff in their countries skilled in assessing VHF cases/outbreaks; 19 laboratories emphasised a need for further training, not only for laboratory workers, but also for medical and nursing staff.Half of the 24 laboratories with CCHF diagnostic capacity stated their availability to offer training services for CCHF diagnosis to other laboratories in and outside their countries.
Of all responding laboratories, 20 had standardised procedures for specimen collection and storage of CCHF infected material, and 25 for processing and shipping suspected CCHF specimens for confirmation diagnosis in other laboratories.

Diagnostic capacities
Of the 31 laboratories that participated in the survey, 24 declared to have set up diagnostic capacities to detect CCHF infection.The remaining seven laboratories in countries where CCHF diagnostic capacities has not yet been established, declared that they were sending samples to reference laboratories or WHOCCs outside their countries (Figure 1).
Among the 24 laboratories with diagnostic capacities, all except the laboratory in Serbia had CCHF molecular tests based on either quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) or nested RT-PCR.Information on the type of commercial or in-house protocol used was requested from the participants.Among the 23 laboratories which performed CCHF molecular diagnosis, 20 used an inhouse method, 11 used commercial assays and eight combined both in-house and commercial approaches (Figure 2).3).

Biosafety
The 24 laboratories with diagnostic capacities informed about the inactivation process before handling specimens for diagnostic purpose.Among them, six laboratories inactivated specimens under BSL4, 12 in BSL3 and five in BSL2 conditions (Figure 3).Of 11 laboratories performing viral isolation and propagation, six did so in BSL4 facilities and five in lower-grade BSL facilities.

Discussion
This survey has been carried out in 28 countries of the European region, including 10 countries where human cases are frequently or sporadically reported, or where there has been evidence of CCHFV circulation in animals or ticks.The presence of potential CCHFV vectors in other European countries may extend the current geographical distribution of the disease.In addition, imported cases in travellers have been reported in the EU.Hence, early recognition of the suspected CCHF cases is critical, in order to initiate the proper treatment of the patient and to apply control measures for containment of the disease.Some authors argue that Europe needs to implement a harmonised case definition for CCHF in order to enhance notifications and to estimate the diseases burden and epidemiological trends in various areas and countries [2].The survey revealed that all responding countries used the generic case definition of VHFs to identify and notify CCHF cases.However, this survey has some limitations since not all responding countries clearly specified the source and reference of the generic or specific case definitions.
Networking and training are key factors in ensuring a rapid and effective response to CCHF.The survey revealed that the majority of countries belong to at least one network apart from ENIVD that could assure support, management, training in the diagnosis of CCHF cases, expert consultation, exchange of experiences and protocols, and scientific support if needed.Considering that some respondents did not have procedures in place for specimen collection, processing or transporting, the networks could also play a key role in closing this gap.The networks could also foster training via organising international workshops on CCHF diagnosis and biosafety.
Laboratory techniques are the cornerstone of CCHF diagnosis, essential for effective surveillance, management of individual patients and outbreak prevention.
In 2008, the multidisciplinary consultation of CCHF experts organised by ECDC showed that according to ENIVD, 15 of 27 countries performed CCHF diagnostics [13].The current survey launched in 2012 indicated an increase to 21 of 28 countries performing CCHF diagnostics.Our results show a strong increase in the diagnostic capacity for CCHF from 2008 to the present, possibly due to the nomination of CCHF as a priority disease for the EU.However, as shown in Table 1, two WHOCC next to endemic areas (Greece and Slovenia), lost their status as reference centres for VHF.This issue has to be taken in consideration when a new reference centre in Europe will be designated in the future.
Currently, the routine laboratory diagnosis of CCHF is based mainly on the detection of the viral genome and specific IgM and IgG.Most surveyed laboratories with diagnostic capacities (21/24) followed international recommendations of combining molecular and serological methods for CCHF diagnosis [1,28].This shows that most of the surveyed laboratories have essential diagnostic tools for CCHF diagnosis in place.
Molecular assays offer a rapid, sensitive and specific diagnosis of CCHF during the viraemic phase of infection up to day 16 of illness [29].The vast majority of surveyed countries (20/21) have molecular tests available, and most of them participated in CCHF EQAs.It is highly recommended that not only endemic countries, but also neighbouring countries that lack the capacity for molecular assays try to implement them.
Of the existing molecular methods for CCHF diagnosis, the majority of respondents (18/20) used a qRT-PCR, combined or not with nested PCR, while the remaining two countries used a nested RT-PCR only.Moreover, in a recent molecular EQA, it is reported that nested RT-PCR performs considerably less well compared with qRT-PCRs [16].Therefore, it is recommended that countries performing only nested RT-PCR implement capacities for a quantitative assay because qRT-PCRs offer advantages when over nested RT-PCR such as lower contamination rate, higher sensitivity and specificity, and better time-effectiveness.A factor that may limit the use of molecular diagnostic methods is the fact that sensitivity may be affected by the high diversity of CCHF genomes.For instance, it has been found that sensitivity of molecular methods was associated with the patients' country of origin [17].A combination of commercial and in-house RT-PCR assays will probably ensure the detection of CCHFV strains despite their diversity.However, the survey reveals that 20 of 23 laboratories use in-house RT-PCR but only eight combine it with a commercial test.
Although serological methods may cover a broader spectrum of strains due to cross-reactivity, attention must be also paid to antigenic variation among CCHFV strains which may affect their sensitivity.However, combinations of ELISA and IFA, commercial or in-house, may increase the sensitivity of detection.A recent evaluation of two commercial kits (VectorBest ELISAs and Euroinmune IFA, both for IgM and IgG) revealed that efficient and well characterised serological assays and protocols are available for CCHF diagnosis [17].Our survey reveals that all countries using the commercial ELISA also had available commercial IFAs and that half of them combined them with an in-house ELISAs that may compensate a potential lower sensitivity caused by antigenic diversity.We advise that each country assure that their methods are optimised for strains circulating in their area, or that they use an adapted method for CCHFV genotypes circulating in their country.
In addition, to assure that diagnostic methods perform with optimal accuracy, an increased effort is needed to establish EQA studies on a regular basis.In 2011, an international EQA for the molecular detection of CCHF was launched [16].The majority of countries with areas endemic for or at risk of CCHF surveyed in our study  In conclusion, the main priority issues to be addresses by European health authorities are: (i) establishing rapid and reliable protocols for CCHF laboratory diagnosis together with guidelines on storage, processing and transportation of samples, (ii) nominating a Regional Reference Expert Laboratory or a WHOCC in or near the endemic areas, and (iii) a comprehensive review of the BSL facilities suited to the reality in the endemic areas, their capacities and capabilities.

Figure 1
Figure 1 Diagnostic capacities and occurrence of Crimean-Congo haemorrhagic fever in Europe since 2000

Table 1
[16,17]urvey on Crimean-Congo haemorrhagic fever, responding laboratories, by country, 2012 (n=31) (EQA) for CCHF molecular diagnosis were carried out to monitor and compare the performance of the different techniques applied for diagnosis of CCHF[16,17].The current situation with continous high transmission in Turkey and south-western Russia, new imported cases in the European Union (EU), detection of the virus for the first time in the western Mediterranean region,MethodsTo gather information on CCHF diagnostics, preparedness and response capacities in Europe, a questionnaire was developed and sent electronically in January 2012 to laboratory contact points in the ENIVD database, covering 28 Member States of the EU as well as nine non-EU countries, Russia, Norway, Switzerland, Bosnia and Herzegovina, Serbia, Kosovo under UN Security Council Resolution 1244, Albania, the Former Yugoslavia Republic of Macedonia and Turkey.All Vector-Best, Novosibirsk, Russia) ELISA tests for CCHF as well as in-house or commercial (EuroImmun, Luebeck, Germany) IFA (Table2).
The serological diagnosis of CCHFV infection is based on the detection of specific IgM and IgG antibodies against recombinant nucleoprotein as the predominant available antigen, either in an enzyme-linked immunosorbent assay (ELISA) or in an indirect immunofluorescence assay (IFA).Most laboratories(22/24)with diagnostic capacities had available at least one serological technique, ELISA or IFA.Respondents were also asked about the availability of specific in-house or commercial (

Table 3
Laboratory preparedness and response capacities for Crimean-Congo haemorrhagic fever diagnosis in the European region, ENIVD survey, 2012 (n=28 countries) Network for Capacity Building for the Control of Emerging Viral Vector Borne Zoonotic Diseases; BSL: biosafety level; CCH-FEVER: Crimean Congo Haemorrhagic Fever Network; ECDC: European Centre for Disease Prevention and Control; ELISA: enzyme-linked immunosorbent assay; ENIVD: European Network for Diagnostics of 'Imported' Viral Diseases; EpiSouth: Network for Communicable Disease Control in Southern Europe and Mediterranean Countries; Euronet-P4: European Network of Biosafety-Level-4 laboratories; EQA: external quality assessment; IFA: indirect immunofluorescence assay; NA: not available; PCR: polymerase chain reaction; QUANDHIP: Quality Assurance exercise and Networking on the Detection of Highly Infectious Pathogens project; VHF: viral haemorrhagic fever; VI: viral isolation.The most definite way of CCHF diagnosis is detection of viral RNA combined with detection of IgM antibodies.Virus isolation as a diagnostic tool is rarely applied because high biocontainment laboratories (BSL4) are required.None of the European BSL4 laboratories are situated in CCHF areas, and among 11 laboratories performing viral propagation, five reported that they do not work in BSL4 facilities.Three of these five laboratories were in CCHF endemic countries.