Molecular monitoring of insecticide resistance in major disease vectors in Armenia.

BACKGROUND: Armenia is considered particularly vulnerable to life-threatening vector-borne diseases (VBDs) including malaria, West Nile virus disease and leishmaniasis. However, information relevant for the control of the vectors of these diseases, such as their insecticide resistance profile, is scarce. The present study was conducted to provide the first evidence on insecticide resistance mechanisms circulating in major mosquito and sand fly populations in Armenia. METHODS: Sampling sites were targeted based mainly on previous historical records of VBD occurrences in humans and vertebrate hosts. Initially, molecular species identification on the collected vector samples was performed. Subsequently, molecular diagnostic assays [polymerase chain reaction (PCR), Sanger sequencing, PCR-restriction fragment length polymorphism (RFLP), quantitative PCR (qPCR)] were performed to profile for major insecticide resistance mechanisms, i.e. target site insensitivity in voltage-gated sodium channel (vgsc) associated with pyrethroid resistance, acetylcholinesterase (ace-1) target site mutations linked to organophosphate (OP) and carbamate (CRB) resistance, chitin synthase (chs-1) target site mutations associated with diflubenzuron (DFB) resistance and gene amplification of carboxylesterases (CCEs) associated with resistance to the OP temephos. RESULTS: Anopheles mosquitoes were principally represented by Anopheles sacharovi, a well-known malaria vector in Armenia, which showed no signs of resistance mechanisms. Contrarily, the knockdown resistance (kdr) mutations V1016G and L1014F/C in the vgsc gene were detected in the arboviral mosquito vectors Aedes albopictus and Culex pipiens, respectively. The kdr mutation L1014S was also detected in the sand fly, vectors of leishmaniasis, Phlebotomus papatasi and P. tobbi, whereas no mutations were found in the remaining collected sand fly species, P. sergenti, P. perfiliewi and P. caucasicus. CONCLUSIONS: This is the first study to report on molecular mechanisms of insecticide resistance circulating in major mosquito and sand fly disease vectors in Armenia and highlights the need for the establishment of systematic resistance monitoring practices for the implementation of evidence-based control applications.

Assessing the Adoption of One Health Approaches in National Plans to Combat Health Threats: The Pilot of a One Health Conceptual Framework in Armenia.

Due to several factors, such as environmental and climate changes, the risk of health threats originating at the human-animal-environment interface, including vector-borne diseases (VBDs) and zoonoses, is increasing. Low-resource settings struggle to counter these multidimensional risks due to their already-strained health systems and are therefore disproportionally affected by the impact caused by these changes. Systemic approaches like One Health (OH) are sought to strengthen prevention and preparedness strategies by addressing the drivers of potential threats with a multidisciplinary and multisectoral approach, considering the whole system at the human-animal-environment interface. The integration of OH in national plans can be challenging due to the lack of effective coordination and collaboration among different sectors. To support the process of knowledge coproduction about the level of OH integration in prevention and preparedness strategies against health threats in Armenia, a situation analysis was performed on Crimean-Congo hemorrhagic fever/virus and anthrax (identified by local stakeholders as priorities to be addressed with the OH approach), and actions to strengthen the national OH system were identified with the support of a OH conceptual framework. The study highlighted that multidisciplinary and multisectoral efforts towards prevention and preparedness against VBDs and zoonoses threats need to be strengthened in Armenia, and priority actions to integrate the OH approach were identified.

Gamasid Ticks as Vectors of Tularemia in the Southeast of Armenia.

Background: The natural environment of southeastern Armenia, which includes the Syunik and Vayots Dzor regions, provides a high biodiversity of flora and fauna, including ectoparasites. Currently, the fauna and ecology of gamasid ticks and their role in the circulation of tularemia in this area are unclear and incomplete. To better understand the persistence of tularemia in Armenia, an assessment of specific hosts and their vectors is needed to evaluate their role in perpetuating tularemia. Materials and Methods: Utilizing data and samples collected from 1970 to 2020, we have evaluated the species composition of gamasid ticks found on the common vole and in their nests and burrows, and identified the presence of tularemia over time. We evaluated five different geographical landscapes: semidesert, dry mountain steppe, mountain steppe, mountain forest, and high mountain in the communities and open areas of Kapan, Goris, Sisian, Meghri, and Jermuk. Results: We determined the density of gamasid ticks in southeastern Armenia over the 50-year period and isolated 20 cultures of tularemia in 12 separate years. Conclusions: It is important to regularly monitor gamasid ticks in southeastern Armenia to clarify the risk factors for the occurrence of tularemia epizootics, among both carriers and vectors, to better understand the full epidemiological picture.

Assessment of expertise in morphological identification of mosquito species (Diptera, Culicidae) using photomicrographs.

Accurate identification of insect species is an indispensable and challenging requirement for every entomologist, particularly if the species is involved in disease outbreaks. The European MediLabSecure project designed an identification (ID) exercise available to any willing participant with the aim of assessing and improving knowledge in mosquito taxonomy. The exercise was based on high-definition photomicrographs of mosquitoes (26 adult females and 12 larvae) collected from the western Palaearctic. Sixty-five responses from Europe, North Africa and the Middle East were usable. The study demonstrated that the responders were better at identifying females (82% correct responses) than larvae (63%). When the responders reported that they were sure of the accuracy of their ID, the success rate of ID increased (92% for females and 88% for larvae). The top three tools used for ID were MosKeyTool (72% of responders), the ID key following Becker et al. [2010. Mosquitoes and their control, 2nd edn. Berlin: Springer] (38%), and the CD-ROM of Schaffner et al. [2001. Les moustiques d'Europe: logiciel d'identification et d'enseignement - The mosquitoes of Europe: an identification and training programme. Montpellier: IRD; EID] (32%), while other tools were used by less than 10% of responders. Responders reporting the identification of mosquitoes using the MosKeyTool were significantly better (80% correct responses) than non-MosKeyTool users (69%). Most responders (63%) used more than one ID tool. The feedback from responders in this study was positive, with the exercise being perceived as halfway between educational training and a fun quiz. It raised the importance of further expanding training in mosquito ID for better preparedness of mosquito surveillance and control programmes.

Title: Évaluation de l'expertise en identification morphologique des espèces de moustiques (Diptera, Culicidae) à l'aide de photomicrographies. Abstract: L'identification précise des espèces d'insectes est une exigence indispensable et difficile pour tout entomologiste, en particulier si l'espèce est impliquée dans des épidémies. Le projet européen MediLabSecure a conçu un exercice d'identification (ID) accessible à tout participant volontaire dans le but d'évaluer et d'améliorer les connaissances en taxonomie des moustiques. L'exercice était basé sur des photomicrographies haute définition de moustiques (26 femelles adultes et 12 larves) prélevées dans le Paléarctique occidental. Soixante-cinq réponses d'Europe, d'Afrique du Nord et du Moyen-Orient ont été utilisables. L'étude a démontré que les répondants étaient meilleurs pour identifier les femelles (82 % de réponses correctes) que les larves (63 %). Lorsque les répondants ont déclaré être sûrs de l'exactitude de leur ID, le taux de réussite de l'identification était meilleur (92 % pour les femelles et 88 % pour les larves). Les trois principaux outils utilisés pour les ID étaient MosKeyTool (72 % des répondants), la clé d'identification du livre de Becker et al. (38%) et le CD-ROM de Schaffner et al. (32 %), tandis que d'autres outils étaient utilisés par moins de 10 % des répondants. Les répondants déclarant identifier des moustiques à l'aide de MosKeyTool étaient significativement meilleurs (80 % de réponses correctes) que les non-utilisateurs de MosKeyTool (69 %). La plupart des répondants (63 %) ont utilisé plus d'un outil d'identification. Les commentaires des répondants de cette étude ont été positifs, l'exercice étant perçu comme à mi-chemin entre une formation pédagogique et un quiz amusant. Il a souligné l'importance d'étendre la formation complémentaire à l'identification des moustiques pour une meilleure préparation des programmes de surveillance et de contrôle des moustiques.

A review of human alveolar echinococcosis in the Republic of Armenia from 2008 to 2020.

Background: Since the 90s' Echinococcus multilocularis infection has expanded the geographical area and central-eastern European countries had seen first alveolar echinococcosis (AE) human cases. AE is considered to be a very rare disease in Europe with average incidences of 0.03-0.2/100,000 inhabitants/year. Because of a suitable orography, this study aims to confirm whether there are human AE cases in Armenia, identify areas at risk, and also estimate AE annual incidence. Methods: Retrospective AE case finding was carried out at main multi-profile medical centers equipped with the modern diagnostic means. The medical records of all patients with liver surgery admitted between January 2008 and June 2020 were reviewed. A specific form was developed in EpiInfo v.7.2. Annual national incidence was estimated using population denominators provided by the National Institute of Statistics. Results: Overall, 11 AE cases have been identified. All patients were diagnosed at advanced stages, with subsequent poor prognosis and costly treatment. Confirmation was based on tissue biopsy and medical imaging results. Age ranged from 12 to 58 years with a median of 33 yrs. Patients were from rural communities, mostly in the Gegharkunik region (6 cases, 55%). Annual average incidence of AE was 0.033/100,000 varying between 0.032 in 2008 and 0.1 in 2017. Conclusions: There are human AE cases happening in Armenia since more a decade ago. In the absence of an AE surveillance system, the burden of AE disease is difficult to estimate. Development of national AE guidelines with a case definition should help enforcing registry of all cases, early diagnosis and also clinical and public awareness.

Phlebotomus (Adlerius) simici NITZULESCU, 1931: first record in Austria and phylogenetic relationship with other Adlerius species.

BACKGROUND: Phlebotomine sand flies are the principal vectors of Leishmania spp. (Kinetoplastida: Trypanosomatidae). Information on sand flies in Central Europe is scarce and, to date, in Austria, only Phlebotomus mascittii has been recorded. In 2018 and 2019, entomological surveys were conducted in Austria with the aim to further clarify sand fly distribution and species composition. RESULTS: In 2019, a Ph. simici specimen was trapped in Austria for the first time. Analyses of two commonly used marker genes, cytochrome c oxidase I (coxI) and cytochrome b (cytb), revealed high sequence identity with Ph. simici specimens from North Macedonia and Greece. Phylogenetic analyses showed high intraspecific distances within Ph. simici, thereby dividing this species into three lineages: one each from Europe, Turkey and Israel. Low interspecific distances between Ph. simici, Ph. brevis and an as yet unidentified Adlerius sp. from Turkey and Armenia highlight how challenging molecular identification within the Adlerius complex can be, even when standard marker genes are applied. CONCLUSION: To our knowledge, this study reports the first finding of Ph. simici in Austria, representing the northernmost recording of this species to date. Moreover, it reveals valuable insights into the phylogenetic relationships among species within the subgenus Adlerius. Phlebotomus simici is a suspected vector of L. infantum and therefore of medical and veterinary importance. Potential sand fly expansion in Central Europe due to climatic change and the increasing import of Leishmania-infected dogs from endemic areas support the need for further studies on sand fly distribution in Austria and Central Europe in general.

The mosquitoes of Armenia: review of knowledge and results of a field survey with first report of Aedes albopictus.

BACKGROUND: In 2016, a field study was implemented in all Armenian provinces in order to update knowledge on the presence and distribution of both native and invasive mosquito species. Larvae and adult mosquitoes were sampled and identified on the basis of their morphology. Supplementary field surveys were performed in 2017-2018. RESULTS: Between June 20 and July 12, 2016, 117 localities were visited. A total number of 197 sampling units were checked, of which 143 (73%) were positive for mosquitoes (with 1-6 species per sampling unit). A total number of 4157 mosquito specimens were identified to species or species complex level. Ten species represent first records for Armenia: Aedes albopictus, Ae. annulipes, Ae. cataphylla, Ae. cinereus/geminus (probably Ae. cinereus), Ae. flavescens, Anopheles plumbeus, Coquillettidia richiardii, Culex martinii, Cx. torrentium and Culiseta subochrea. The invasive species Ae. albopictus was recorded in a single locality (Bagratashen) at the border point with Georgia, along the main road Tbilisi-Yerevan. This species was further recorded in 2017 and 2018, demonstrating its establishment and spread in north Armenia. These surveys confirm the presence of vectors of malaria parasites (in particular An. sacharovi) and West Nile virus (Cx. pipiens). CONCLUSION: The knowledge of the Armenian mosquito fauna is extended to a list of 28 species. The record of Aedes albopictus, an important potential vector of many arboviruses, has important implications for public health.

TITLE: Les moustiques d'Arménie : synthèse des connaissances et résultats d'une étude de terrain avec une première mention pour Aedes albopictus. ABSTRACT: Contexte : En 2016, nous avons réalisé une étude sur le terrain dans toutes les provinces du pays dans le but d'actualiser la présence et la distribution des espèces de moustiques aussi bien natives qu'invasives. Les moustiques récoltés aux stades larvaires et adultes ont été identifiés sur des critères morphologiques. Des suivis additionnels ont été réalisés en 2017 et 2018. Résultats : Entre le 20 juin et le 12 juillet 2016, 117 localités ont été visitées. Au total, 197 unités de collecte ont été prospectées dont 143 (73%) se sont révélées positives pour les moustiques (avec de 1 à 6 espèces par unité de collecte). Au total, 4157 spécimens ont été identifiés au niveau de l'espèce ou du complexe d'espèces. Dix espèces ont été observées pour la première fois en Arménie : Aedes albopictus, Ae. annulipes, Ae. cataphylla, Ae. cinereus/geminus (probablement Ae. cinereus), Ae. flavescens, Anopheles plumbeus, Coquillettidia richiardii, Culex martinii, Cx. torrentium et Culiseta subochrea. L'espèce invasive Ae. albopictus a été observée dans une seule localité (Bagratashen) à la frontière avec la Géorgie, sur l'axe routier principal Tbilisi-Yerevan. Cette espèce a également été observée en 2017 et en 2018, faisant la preuve de son installation et de sa diffusion dans le nord de l'Arménie. Ces suivis confirment la présence des vecteurs de Plasmodium humains (en particulier An. sacharovi) et du virus West Nile (Cx. pipiens). Conclusion : La connaissance de la faune culicidienne d'Arménie progresse, avec une liste actualisée à 28 espèces. L'observation d'Aedes albopictus, un important vecteur potentiel de nombreux arbovirus, a d'importantes implications en termes de santé publique.

Towards harmonisation of entomological surveillance in the Mediterranean area.

BACKGROUND: The Mediterranean Basin is historically a hotspot for trade, transport, and migration. As a result, countries surrounding the Mediterranean Sea share common public health threats. Among them are vector-borne diseases, and in particular, mosquito-borne viral diseases are prime candidates as (re)emerging diseases and are likely to spread across the area. Improving preparedness and response capacities to these threats at the regional level is therefore a major issue. The implementation of entomological surveillance is, in particular, of utmost importance. Guidance in designing entomological surveillance systems is critical, and these systems may pursue different specific objectives depending on the disease. The purpose of the proposed review is to draw up guidelines for designing effective and sustainable entomological surveillance systems in order to improve preparedness and response. However, we make it clear that there is no universal surveillance system, so the thinking behind harmonisation is to define evidence-based standards in order to promote best practises, identify the most appropriate surveillance activities, and optimise the use of resources. Such guidance is aimed at policymakers and diverse stakeholders and is intended to be used as a framework for the implementation of entomological surveillance programmes. It will also be useful to collaborate and share information with health professionals involved in other areas of disease surveillance. Medical entomologists and vector control professionals will be able to refer to this report to advocate for tailored entomological surveillance strategies. The main threats targeted in this review are the vectors of dengue virus, chikungunya virus, Zika virus, West Nile virus, and Rift Valley fever virus. The vectors of all these arboviruses are mosquitoes. METHODS: Current knowledge on vector surveillance in the Mediterranean area is reviewed. The analysis was carried out by a collaboration of the medical entomology experts in the region, all of whom belong to the MediLabSecure network, which is currently funded by the European Union and represents an international effort encompassing 19 countries in the Mediterranean and Black Sea region. FINDINGS: Robust surveillance systems are required to address the globalisation of emerging arboviruses. The prevention and management of mosquito-borne viral diseases must be addressed in the prism of a One Health strategy that includes entomological surveillance as an integral part of the policy. Entomological surveillance systems should be designed according to the entomological and epidemiological context and must have well-defined objectives in order to effect a tailored and graduated response. We therefore rely on different scenarios according to different entomological and epidemiological contexts and set out detailed objectives of surveillance. The development of multidisciplinary networks involving both academics and public authorities will provide resources to address these health challenges by promoting good practises in surveillance (identification of surveillance aims, design of surveillance systems, data collection, dissemination of surveillance results, evaluation of surveillance activities) and through the sharing of effective knowledge and information. These networks will also contribute to capacity building and stronger collaborations between sectors at both the local and regional levels. Finally, concrete guidance is offered on the vector of the main arbovirus based on the current situation in the area.

Identification of mosquitoes (Diptera: Culicidae): an external quality assessment of medical entomology laboratories in the MediLabSecure Network.

BACKGROUND: Identification of vectors is of prime importance in the field of medical entomology for both operational and research purposes. An external quality assessment of mosquito identification capacities was carried out within the MediLabSecure Network, which is composed of laboratories located in 19 countries close to the European Union around the Mediterranean and Black seas. METHODS: A set of blind samples consisting of 7 or 8 adult mosquitoes and 4 larvae was given to each participant laboratory. In all, 138 adult mosquitoes and 76 larvae of different species were distributed for genus and species identification. RESULTS: All identifications were exclusively morphology based. Overall, 81% of identifications were correct at the genus level, 64% at the species level. The results were highly varied among the 19 participating laboratories. The levels of correct identifications were: 100% (three laboratories), 90-95% (four laboratories), 50-75% (six laboratories) and < 50% (six laboratories). CONCLUSIONS: This evaluation showed the need to maintain efforts in capacity building and quality control in the field of medical entomology and, more specifically, in the morphological identification of the Culicidae.

Surveillance of Arthropod-Borne Viruses and Their Vectors in the Mediterranean and Black Sea Regions Within the MediLabSecure Network.

PURPOSE OF REVIEW: Arboviruses, viruses transmitted by arthropods such as mosquitoes, ticks, sandflies, and fleas are a significant threat to public health because of their epidemic and zoonotic potential. The geographical distribution of mosquito-borne diseases such as West Nile (WN), Rift Valley fever (RVF), Dengue, Chikungunya, and Zika has expanded over the last decades. Countries of the Mediterranean and Black Sea regions are not spared. Outbreaks of WN are repeatedly reported in the Mediterranean basin. Human cases of RVF were reported at the southern borders of the Maghreb region. For this reason, establishing the basis for the research to understand the potential for the future emergence of these and other arboviruses and their expansion into new geographic areas became a public health priority. In this context, the European network "MediLabSecure" gathering laboratories in 19 non-EU countries from the Mediterranean and Black Sea regions seeks to improve the surveillance (of animals, humans, and vectors) by reinforcing capacity building and harmonizing national surveillance systems to address this important human and veterinary health issue. The aim of this review is to give an exhaustive overview of arboviruses and their vectors in the region. RECENT FINDINGS: The data presented underline the importance of surveillance in the implementation of more adapted control strategies to combat vector-borne diseases. Partner laboratories within the MediLabSecure network present a wide range of infrastructures and have benefited from different training programs. SUMMARY: Although reporting of arboviral presence is not carried out in a systematic manner, the expansion of the area where arboviruses are present cannot be disputed. This reinforces the need for increasing surveillance capacity building in this region to prevent future emergences.

A retrospective chart review study to describe selected zoonotic and arboviral etiologies in hospitalized febrile patients in the Republic of Armenia.

BACKGROUND: Scant information is available on the infectious causes of febrile illnesses in Armenia. The goal of this study was to describe the most common causes, with a focus on zoonotic and arboviral infections and related epidemiological and clinical patterns for hospitalized patients with febrile illnesses of infectious origin admitted to Nork Infectious Diseases Clinical Hospital, the referral center for infectious diseases in the capital city, Yerevan. METHOD: A chart review study was conducted in 2014. Data were abstracted from medical charts of adults (≥18 years) with a fever (≥38 °C), who were hospitalized (for ≥24 h) in 2010-2012. RESULTS: Of the 600 patients whose charts were analyzed, 76 % were from Yerevan and 51 % were male; the mean age (± standard deviation) was 35.5 (±16) years. Livestock exposure was recorded in 5 % of charts. Consumption of undercooked meat and unpasteurized dairy products were reported in 11 and 8 % of charts, respectively. Intestinal infections (51 %) were the most frequently reported final medical diagnoses, followed by diseases of the respiratory system (11 %), infectious mononucleosis (9.5 %), chickenpox (8.3 %), brucellosis (8.3 %), viral hepatitis (3.2 %), and erysipelas (1.5 %). Reviewed medical charts included two cases of fever of unknown origin (FUO), two cutaneous anthrax cases, two leptospirosis cases, three imported malaria cases, one case of rickettsiosis, and one case of rabies. Engagement in agricultural activities, exposure to animals, consumption of raw or unpasteurized milk, and male gender were significantly associated with brucellosis. CONCLUSION: Our analysis indicated that brucellosis was the most frequently reported zoonotic disease among hospitalized febrile patients. Overall, these study results suggest that zoonotic and arboviral infections were not common etiologies among febrile adult patients admitted to the Nork Infectious Diseases Clinical Hospital in Armenia.

Intestinal Infections Among Febrile Hospitalized Patients in the Republic of Armenia: A Retrospective Chart Review.

In the past, several enteric outbreaks in 1996, 1998, 1999, and 2003 caused by Salmonella typhi, a Gram-negative bacterium, have occurred in Armenia. This study describes the demographic, epidemiological, and clinical characteristics of febrile hospitalized patients with intestinal infections in Armenia. Using a chart review study design, medical data from adult patients who were hospitalized at the Nork hospital during 2010-2012 were reviewed. A total of 600 medical charts were reviewed. Of these, 51 % were diagnosed with intestinal infections. Among these patients, 59 % had an intestinal infection of known etiology, with three main pathogens identified: Salmonella sp. (32 %), Shigella sp. (32 %), and Staphylococcus aureus (18 %). After controlling for the calendar year, age in years, and gender, patients detected with Salmonella sp. were more likely to reported the presence of a family member with similar signs or symptoms [odds ratio (OR) 9.0; 95 % CI 2.4-33.7] and the lack of a water tap at home (OR 3.9; 95 % CI 1.7-9.5). Evidence indicates that Salmonella sp., Shigella sp., and S. aureus as the most common etiologies reported among febrile hospitalized patients. A high percentage of patients had intestinal infections of unknown etiology; thus, improvement in laboratory capacity (enabling more advanced tests, such as polymerase chain reaction) would increase the identification of the enteropathogens causing disease in Armenia.