key: cord-0795582-iqgs1q81 authors: Mikaty, Guillain; Mathéus, Séverine; Mantke, Oliver Donoso; McCulloch, Elaine; Zeichhardt, Heinz; Manuguerra, Jean-Claude title: Evaluation of the diagnostic capacities for emerging arboviral diseases in the international network MediLabSecure from 2014 to 2018 - Importance of External Quality Assessments date: 2021-12-09 journal: J Infect Public Health DOI: 10.1016/j.jiph.2021.12.005 sha: 67b5e5c6c304f37df3897dbcb078161224d7d09c doc_id: 795582 cord_uid: iqgs1q81 BACKGROUND: Emerging infectious diseases pose an increasing threat to all nations around the world, including to developed countries. By definition, because they are rare or unknown, public health systems are not well prepared against these emerging diseases. To be fully prepared, countries must have implemented surveillance systems to monitor rare or unusual sanitary events. METHODS: The capacity of diagnostic laboratories is a key component of surveillance systems since they are in charge of identifying the pathogens responsible for outbreaks in a timely manner. The MediLabSecure project aims at implementing a comprehensive surveillance system for vector-borne diseases around the Mediterranean and Black Sea regions. From 2014 to 2018, the human-virology group of MediLabSecure notably supported the implementation of molecular diagnostic capacities for eight arboviruses and one coronavirus in 19 laboratories of its network through sharing of protocols and reagents, and technical training of the scientific staff of beneficiary laboratories. RESULTS: We report the results of External Quality Assessments for four of these viruses to assess the efficiency of the diagnostic for these threats emerging in the geographic area. The results for these EQA demonstrate the success of the project in the implementation of diagnostic technics for the identification of Dengue, Chikungunya, Zika, and West Niles viruses in laboratories that did not have the capacity before. However, results also show that some work is still to be done to strengthen the newly acquired capacity. CONCLUSION: The MediLabSecure project deployed an effort to build an efficient capacity in identifying and survey the emergence of arboviruses in the Mediterranean area. Diagnostic technics were successfully implemented in many of the laboratories of the network, but the effort must be maintained over time to strengthen these capacities. Over the early twenty-first century, emerging infectious diseases became a major concern for public health in numerous countries. In 2018 and most recently in 2020, WHO updated the list of priority diseases for research and development [1] . This list now includes mainly emerging diseases caused by arboviruses, such as Rift Valley fever (RVF) or Crimean-Congo haemorrhagic fever (CCHF) viruses; zoonotic haemorrhagic or encephalitic viruses, i.e. Ebola, Lassa, or Nipah viruses; and the zoonotic coronaviruses responsible for lower respiratory infections and acute respiratory distress syndromes, i.e. MERS-CoV (cause of the Middle East respiratory syndrome, MERS), SARS-CoV-1 (associated with severe acute respiratory syndrome, SARS), and SARS-CoV-2 (currently causing the ongoing COVID-19 pandemic). The capacity of a country to respond quickly to the emergence of a pathogen starts at the laboratory level with the detection of the first cases. This detection is key in controlling the resulting epidemic. In 2013, failure to detect Zika virus (ZIKV) in the South Pacific and in South America resulted in missing the rapid increase in cases and led to a one-year delay in the international response [2] . In 2014, in Guinea, the inability to identify Ebola virus, which was unknown in this region, led to a four-month delay in the response to control the epidemic, partly responsible for the magnitude of the Ebola epidemic [3] . The MediLabSecure project is an international project funded by the European Commission since 2014. Its purpose is to enhance preparedness in preventing and controlling the emergence of zoonotic viruses around the Mediterranean Sea, the Black Sea, and since 2018, the Sahel It represents a cluster for awareness, risk assessment, surveillance, monitoring, and control of relevant emerging diseases, with a special focus on arboviral infections. MediLabSecure operates at different levels: (i) At the laboratory level, MediLabSecure implements and/or improves pathogen detection [5] , with a particular emphasis on biosafety and biosecurity processes; (ii) at the institutional level, the project advocates integrated surveillance, One Health collaborations, and tries to support the establishment of early warning systems in countries [6, 7] ; (iii) at the international level, MediLabSecure promotes networking to enhance collaborations among neighbouring countries. To evaluate the capabilities and guarantee the quality of laboratory results, the MediLabSecure project sponsored the participation of the beneficiary laboratories to a series of EQA programmes. Two non-profit EQA providers were engaged for the organization of molecular Quality Control for Molecular Diagnostics (QCMD) [10] , an independent international EQA and Proficiency Testing (PT) organization, proposed four EQA schemes to assess the proficiency of laboratories in the detection of WNV (incl. optional lineage typing), CHIKV, DENV (incl. optional type identification), and ZIKV in reconstituted biological samples. Positive samples (for WNV, CHIKV, DENV, and ZIKV) were obtained from supernatants of virus-infected cell cultures and inactivated afterwards by heat and gamma-irradiation, while samples with other cell culture-derived viruses were used as specificity controls or samples with transport medium only were used as true negative controls. The EQA providers do not refer to concentrations of samples in qualitative EQA schemes. Only the dilution ratios or the sample relationships by dilution series and order of reducing titer are used in the reports of the respective EQA providers to indicate sensitivities based on low, medium, or high diluted samples. The actual values of titers (high, medium, or low) depend on the virus considered to reflect real biological samples. For this report, we kept the providers' formal presentations of results. Nineteen laboratories from nineteen countries participated within the human-virology laboratory network during 2014 to 2018 (see Supplemental Figure 1 ). A questionnaire sent during the selection process allowed to identify the needs and gaps of the newly created network. It also served as basis for the capacity building strategy developed during the following years. Regarding laboratory capabilities for the identification of the viruses targeted within the project, the responses to the questionnaire showed that 79% of the laboratories declared capabilities for the molecular diagnostic for WNV, while 63% implemented DENV diagnostic (see Supplemental Figure 2 ). However, for other arboviruses, the declared capabilities were much lower with 37% for CCHFV, 32% for tick-borne encephalitis virus (TBEV), 21% for CHIKV, 16% for RVFV infections, and only 5% (1 laboratory) for YFV. Of (Table 2 ). Despite globally satisfactory results for the identification of both WNV and CHIKV in 2016, the network still presented with room for improvement. In 2018, MediLabSecure proposed to J o u r n a l P r e -p r o o f all human-virology laboratories to participate in EQA programmes from QCMD regarding some of the viral targets of the project: WNV, CHIKV, ZIKV, and DENV. Others as MERS-CoV EQA were performed but not reported here. Beneficiary laboratories were free to register for some or all programmes. The nineteen laboratories responded, and all of them registered to at least one of the EQAs, often to the four. Unfortunately, some laboratories were not capable to answer in a timely manner. Each responding laboratory sent one dataset to QCMD. Altogether, an average of 74.3% of laboratories that registered participated in the EQAs and In 2018, the human-virology laboratories registered to a second EQA programme on WNV conducted by QCMD ( Table 3 ). The coordinating laboratory of the human-virology network participated in this EQA. Overall, 60% (9/15) of the laboratories successfully identified all the core samples of the programme and validated the EQA. This rate was equivalent to the one measured two years before, suggesting stability but no improvement. However, individually, at least 93.3% (14/15) of the laboratories correctly identified each sample with the notable exception of the two lower-concentrated WNV lineage 1 (WNV NY99) samples, MEDWNV18-02 (80% success (12/15)) and MEDWNV18-07 (66.7% success (10/15)). This result suggests a lineage specific sensitivity issue for the techniques employed since the lineage 2 (WNV Heja) was correctly identified by 93.3% of laboratories at its lower concentration. The MEDWNV18-07 was considered an educational sample and its correct identification was not necessary to validate the quality programme. The specificity of identification was quite good with at least 93.3% of correct identification for the three negative samples, including two specificity controls with other flaviviruses. Thus, the lower global success rate of 60% was due J o u r n a l P r e -p r o o f to six laboratories doing one misidentification rather than a couple of laboratories with an inadequate technique. [1] Detection Frequency [2] / Sample Status [3] Datasets (n) Success Rate [4] MEDWNV18- The results of the second EQA on CHIKV identification organized by QCMD are displayed in ships [1] Detection Frequency [2] / Sample Status [3] Datasets (n) Rate [4] MEDCHIK18- After the global epidemic of 2013-2016, the molecular detection of this virus was implemented in the region partly thanks to the MediLabSecure project [5] . The QCMD EQA programme [11] was also the opportunity to evaluate the implementation of the techniques in the participating laboratories. The results were quite satisfactory (Table 5 ) and around 64% (9/14) of the laboratories participating in the EQA programme correctly identified all core samples. Individually, five samples were identified with a success rate of 100%, and two samples by 92.9% (13/14) of the participants. As for the other EQAs, the difficulty was met for the lowest concentrated ZIKV African lineage sample ZIKA18S-06 with only 71.4% (10/14) successfully identifying this sample. The specificity was very good since 100% of the laboratories identified the negative samples (including also a specificity control with other flaviviruses). ships [1] Detection Frequency [2] / Sample Status [3] Datasets (n) Rate [4] ZIKA18S-01 ZIKV The results for the QCMD EQA among the participating countries were acceptable despite a moderate global success rate of 61.5% (8/13) in the programme. Individually, five samples were correctly identified by 92.3% (12/13) of the participants and four samples were correctly identified by all participants ( Table 6 ). The educational sample DENVRNA18S-03, with the lowest concentration of DENV-3, was correctly identified by 84.6% (11/13) of the laboratories. The specificity was excellent with 100% identification of the negative samples (including also other flaviviruses as specificity control). The moderate global success rate was due to five different laboratories reporting one mistake each with the core samples. [1] Detection Frequency [2] / Sample Status [3] Datasets (n) Success Rate [4] DENVRNA 18S-01 61.5%* [1] Sample Relationships: Indicates the relationships of the samples within this challenge. The highest titer member of dilution series DS1 is indicated by DS1_1 and further members of the series as DS1_2, DS1_3 etc. in order of reducing titer. Additional dilution series are indicated by DS2 (e.g., DS2_1, DS2_2 etc.). One duplicate pair is present and is indicated by 'D1'. [2] Detection Frequency: To aid qualitative analysis each panel member is assigned a frequency of detection. This is based on the peer group consensus of all qualitative results returned from participants within the EQA challenge / distribution. The MediLabSecure project aims to prevent emerging infectious diseases through multiple and complementary actions in its geographical area. At the laboratory level, the project notably promotes the implementation of detection techniques for a panel of emerging arboviruses and respiratory viruses of zoonotic origin. Indeed, the detection of the pathogens is the first key step to raise awareness and alert authorities about the risk of a particular epidemic. The first action of the MediLabSecure project was to create a network of legitimate sentinel laboratories. The selection of the laboratories for each specialty was key in the process. The EQAs are a common tool to control and guarantee the efficiency and quality of the techniques implemented in diagnostic laboratories and are mandatory for some quality certifications and accreditation. From 2014 to 2018, the MediLabSecure project sponsored the participation of the network laboratories from veterinarian and human-virology sectors to different commercial [11] or house-made EQA programmes [13, 14] to test the diagnostic capabilities on WNV, USUV, ZIKV, CHIKV, DENV, YFV, RVF, and MERS-CoV. Specific EQA for the identification of mosquito vectors was created for medical entomology laboratories from the network [15] . The participation to these EQAs was on voluntary basis and some countries could not, or would not, participate because of certain situations (virus absent from the region, customs or shipment difficulties, local epidemic overwhelming the lab capacities, etc.). In this report, the results that the MediLabSecure human-virology laboratories obtained in six EQA programs were presented to evaluate the implementation of molecular diagnostic technics within the network. However, the actual aim of the EQAs was for each beneficiary laboratory to make its own auto-evaluation and serve as quality control for its laboratory qualifications. with less efficiency, as a group, to identify the limit samples (low concentrated samples). Over the six EQA programmes, it was never possible to associate the sensitivity issues to a specific technic, in-house protocol, or commercial kit. The misidentifications were equally distributed amongst the declared technics, suggesting that the limit of detection for different viruses depends on other factors, such as the type of apparatus used, the training of staff, or the local conditions. This sensitivity issue was sometimes strain-specific (or lineage-specific). The results for the WNV EQA from QCMD (2018) suggested a better sensitivity of the techniques for the lineage 2 (WNV Heja) compared to lineage 1 (NY99), which was not expected since lineage 2 is quite recent in the Mediterranean area compared to the lineage 1 and laboratories were more used to identify the latest. Sensitivity limitation is well known for molecular diagnostic and might be an issue to detect arboviruses in blood samples since the viremia is often low and last only a few days. This also shows that there is still room for improvement in the network and that MediLabSecure will benefit from an additional four years. The specificity of the implemented techniques was very good for the arboviruses. Above 90% of the group, often 100%, were capable of correctly identify the negative samples, even when another close virus was present in the samples. In this work, we used the results obtained from EQA programmes to evaluate the efficiency of the MediLabSecure project to transfer competencies. Overall, the EQA programmes showed that the transfer was done and effective for most of the laboratories considering the diagnostics These valuable data must not be understood as the final goal for the project, neither a success nor a failure, but rather as a temporary picture of the progress made, and work left to be done in the capacity building strategy of MediLabSecure. The repetition of EQAs on WNV and CHIKV in 2016 and 2018 showed a slight progression in the group. An important conclusion that these results highlighted is the fact that a correct and sustainable implementation of a diagnostic technique requires more than sharing protocols and reagents, but also necessitates the training of the staff and regular external quality controls over time. WHO prioritizing diseases list Zika virus in the Americas: Early epidemiological and genetic findings Delayed recognition of Ebola virus disease is associated with longer and larger outbreaks Importance of a One Health approach in advancing global health security and the Sustainable Development Goals Risk of Zika virus transmission in the Euro-Mediterranean area and the added value of building preparedness to arboviral threats from a One Health perspective Strengthening Preparedness for Arbovirus Infections in Mediterranean and Black Sea Countries: A Conceptual Framework to Assess Integrated Surveillance in the Context of the One Health Strategy On Behalf Of The MediLabSecure Working Group null. Integrated Early Warning Surveillance: Achilles' Heel of One Health? Microorganisms Surveillance of Viruses and Their Vectors in the Mediterranean and Black Sea Regions Within the MediLabSecure Network QCMD External Quality Assessment (EQA) for Molecular Diagnostics of Zika Virus: Experiences from an International EQA Programme EPISOUTH External quality assessment of Rift Valley fever diagnosis in 17 veterinary laboratories of the Mediterranean and Black Sea regions Evaluation of West Nile Virus Diagnostic Capacities in Veterinary Laboratories of the Mediterranean and Black Sea Regions Identification of mosquitoes (Diptera: Culicidae): an external quality assessment of medical entomology laboratories in the MediLabSecure Network. Parasit Vectors The authors would like to thank all the members from the MediLabSecure project for their tremendous work and warm collaboration over the years. The MediLabSecure Project is funded by the European Commission through the Instrument contributing to Stability and Peace and the EU CBRN CoE initiative (DG DEVCO IFS/2013/330 961).