A Mark-Release-Recapture Study to Estimate Field Performance of Imported Radio-Sterilized Male Aedes albopictus in Albania.

The pathogen transmitting Aedes albopictus mosquito is spreading rapidly in Europe, putting millions of humans and animals at risk. This species is well-established in Albania since its first detection in 1979. The sterile insect technique (SIT) is increasingly gaining momentum worldwide as a component of area-wide-integrated pest management. However, estimating how the sterile males will perform in the field and the size of target populations is crucial for better decision-making, designing and elaborating appropriate SIT pilot trials, and subsequent large-scale release strategies. A mark-release-recapture (MRR) experiment was carried out in Albania within a highly urbanized area in the city of Tirana. The radio-sterilized adults of Ae. albopictus Albania strain males were transported by plane from Centro Agricoltura Ambiente (CAA) mass-production facility (Bologna, Italy), where they were reared. In Albania, sterile males were sugar-fed, marked with fluorescent powder, and released. The aim of this study was to estimate, under field conditions, their dispersal capacity, probability of daily survival and competitiveness, and the size of the target population. In addition, two adult mosquito collection methods were also evaluated: BG-Sentinel traps baited with BG-Lure and CO2, (BGS) versus human landing catch (HLC). The overall recapture rates did not differ significantly between the two methods (2.36% and 1.57% of the total male released were recaptured respectively by BGS and HLC), suggesting a similar trapping efficiency under these conditions. Sterile males traveled a mean distance of 93.85 ± 42.58 m and dispersed up to 258 m. Moreover, they were observed living in the field up to 15 days after release with an average life expectancy of 4.26 ± 0.80 days. Whether mosquitoes were marked with green, blue, yellow, or pink, released at 3.00 p.m. or 6.00 p.m., there was no significant difference in the recapture, dispersal, and survival rates in the field. The Fried competitiveness index was estimated at 0.28. This mark-release-recapture study provided important data for better decision-making and planning before moving to pilot SIT trials in Albania. Moreover, it also showed that both BG-traps and HLC were successful in monitoring adult mosquitoes and provided similar estimations of the main entomological parameters needed.

Geographic distribution of the V1016G knockdown resistance mutation in Aedes albopictus: a warning bell for Europe.

BACKGROUND: Colonization of large part of Europe by the Asian tiger mosquito Aedes albopictus is causing autochthonous transmission of chikungunya and dengue exotic arboviruses. While pyrethroids are recommended only to reduce/limit transmission, they are widely implemented to reduce biting nuisance and to control agricultural pests, increasing the risk of insurgence of resistance mechanisms. Worryingly, pyrethroid resistance (with mortality < 70%) was recently reported in Ae. albopictus populations from Italy and Spain and associated with the V1016G point mutation in the voltage-sensitive sodium channel gene conferring knockdown resistance (kdr). Genotyping pyrethroid resistance-associated kdr mutations in field mosquito samples represents a powerful approach to detect early signs of resistance without the need for carrying out phenotypic bioassays which require availability of live mosquitoes, dedicated facilities and appropriate expertise. METHODS: Here we report results on the PCR-genotyping of the V1016G mutation in 2530 Ae. albopictus specimens from 69 sampling sites in 19 European countries. RESULTS: The mutation was identified in 12 sites from nine countries (with allele frequencies ranging from 1 to 8%), mostly distributed in two geographical clusters. The western cluster includes Mediterranean coastal sites from Italy, France and Malta as well as single sites from both Spain and Switzerland. The eastern cluster includes sites on both sides of the Black Sea in Bulgaria, Turkey and Georgia as well as one site from Romania. These results are consistent with genomic data showing high connectivity and close genetic relationship among West European populations and a major barrier to gene flow between West European and Balkan populations. CONCLUSIONS: The results of this first effort to map kdr mutations in Ae. albopictus on a continental scale show a widespread presence of the V1016G allele in Europe, although at lower frequencies than those previously reported from Italy. This represents a wake-up call for mosquito surveillance programs in Europe to include PCR-genotyping of pyrethroid resistance alleles, as well as phenotypic resistance assessments, in their routine activities.

Epidemiological and clinical characteristics of imported malaria in Bulgaria: A retrospective study of а 21-year period.

BACKGROUND: Bulgaria, with a high endemicity for malaria in the past, was declared by the WHO as a malaria-free country in 1965. We intended to analyze the epidemiological and clinical implications of imported malaria cases in Bulgaria. METHODS: This is a retrospective cross-sectional analysis of all recorded cases of imported malaria in Bulgaria over a 21-year period (2000-2020). Patients' clinical records and information gathered from the epidemiological survey of each recorded malaria case were reviewed. RESULTS: A total of 232 cases of imported malaria were reported, 147 (63.4%) were Bulgarian citizens (BC) and 85 (36.6%) were foreign nationals (FN). Two thirds (66.4%) of cases were diagnosed from April to October. Most BCs had travelled for work (66.6%) to Africa (93.9%) and were infected with P. falciparum (83.3%), while most FNs were migrants (54.7%), exposed in Asia (63.5%) with P. vivax infection (62.4%). Clinical complications and a fatal outcome were noted in 14.7% (n = 34) and 3.5% (n = 8) of cases respectively. All complicated cases were in BNs with P. falciparum infection. CONCLUSIONS: Bulgaria experiences a steady import of malaria. Efforts to improve diagnosis, management and prevention of malaria, as well as maintenance of a high degree of epidemiological vigilance are needed.

Sand flies (Diptera: Psychodidae) in eight Balkan countries: historical review and region-wide entomological survey.

BACKGROUND: Sand flies (Diptera: Psychodidae) are medically important vectors of human and veterinary disease-causing agents. Among these, the genus Leishmania (Kinetoplastida: Trypanosomatidae), and phleboviruses are of utmost importance. Despite such significance, updated information about sand fly fauna is missing for Balkan countries where both sand flies and autochtonous leishmaniases are historically present and recently re-emerging. Therefore, a review of historical data on sand fly species composition and distribution in the region was followed by a large-scale entomological survey in eight Balkan countries to provide a recent update on local sand fly fauna. METHODS: The literature search involved the period 1910-2019. The entomological survey was conducted at 1189 sampling stations in eight countries (Bulgaria, Bosnia and Herzegovina, Croatia, Kosovo, Montenegro, North Macedonia, Serbia and Slovenia), covering 49 settlements and 358 sampling sites between June and October in the years 2014 and 2016, accumulating 130 sampling days. We performed a total of 1189 trapping nights at these stations using two types of traps (light and CO2 attraction traps) in each location. Sampling was performed with a minimal duration of 6 (Montenegro) and a maximal of 47 days (Serbia) between 0-1000 m.a.s.l. Collected sand flies were morphologically identified. RESULTS: In total, 8490 sand fly specimens were collected. Morphological identification showed presence of 14 species belonging to genera Phlebotomus and Sergentomyia. Historical data were critically reviewed and updated with our recent findings. Six species were identified in Bosnia and Herzegovina (2 new records), 5 in Montenegro (2 new records), 5 in Croatia (2 new records), 9 in Bulgaria (5 new records), 11 in North Macedonia (1 new record), 10 in Serbia (no new records), 9 in Kosovo (3 new records) and 4 in Slovenia (no new records). CONCLUSIONS: This study presents results of the first integrated sand fly fauna survey of such scale for the Balkan region, providing first data on sand fly populations for four countries in the study area and presenting new species records for six countries and updated species lists for all surveyed countries. Our findings demonstrate presence of proven and suspected vectors of several Leishmania species.

West Nile virus lineage 2 in humans and mosquitoes in Bulgaria, 2018-2019.

BACKGROUND: West Nile virus (WNV) lineage 2, and especially the Hungarian clade, predominates in Europe. Most of the Hungarian clade strains cluster into 2 groups: Central/South-West European and Balkan. OBJECTIVES: Since there was not any study on WNV in mosquitoes in Bulgaria, the present study was designed to test Culex spp. mosquitoes in areas near the Danube river. The aim of the study was to gain an insight into the recent molecular epidemiology of WNV in Bulgaria. STUDY DESIGN: A total of 1871 Culex pipiens mosquitoes collected in 2018 and clinical samples from 23 patients with West Nile neuroinavsive disease observed in 2018 and 2019 were tested by TaqMan RT-PCR and RT-nested PCR and PCR products were sequenced. RESULTS: WNV RNA was detected in clinical samples from 10 patients and in five (12.2 %) of 41 pools of Cx. pipiens mosquitos by realtime RT-PCR, resulting in a minimum infection rate of mosquitoes of 0.27 %. Phylogenetic analysis based on partial NS3 gene sequences from one clinical sample and four mosquito pools showed that all sequences clustered into the Hungarian clade of WNV lineage 2 and all but one were identical to respective sequences from Romania. Whole genome sequences of one mosquito pool belong to the Hungarian group of WNV lineage 2 and cluster in a separate subclade from the Bulgarian strain from 2015, suggesting that at least two different introductions occurred in Bulgaria. CONCLUSIONS: The current study provides insights into the geographic distribution of WNV in Bulgaria.

Practical management plan for invasive mosquito species in Europe: I. Asian tiger mosquito (Aedes albopictus).

Aedes albopictus, also known as the "Asian Tiger Mosquito", is an invasive mosquito species to Europe causing high concern in public health due to its severe nuisance and its vectorial capacity for pathogens such as dengue, chikungunya, yellow fever and Zika. Consequently, the responsible authorities implement management activities to reduce its population density, possibly to below noxious and epidemiological thresholds. In urban areas, these aims are difficult to achieve because of the species' ability to develop in a wide range of artificial breeding sites, mainly private properties. This document (Management Plan) has been structured to serve as a comprehensive practical and technical guide for stakeholders in organizing the vector control activities in the best possible way. The current plan includes coordinated actions such as standardized control measures and quality control activities, monitoring protocols, activities for stakeholders and local communities, and an emergency vector control plan to reduce the risk of an epidemic.

An update of the Culicoides (Diptera: Ceratopogonidae) checklist for the Balkans.

BACKGROUND: The prime significance of species belonging to the genus Culicoides Latreille, 1809 (Diptera: Ceratopogonidae) is their ability to transmit viruses such as bluetongue virus (BTV) to wild and domestic ruminants. Prior to 1998, BTV was considered exotic in Europe, but according to recent history of its outbreaks, it has become endemic in southern and eastern European countries circulating beyond its expected historical limits, into the Balkan region. The wind-borne long-distance dispersal of Culicoides spp. over water bodies and local spreading between farms emphasize the necessity of filling in the information gaps regarding vector species distribution. In most Balkan countries, data on Culicoides fauna and species distribution are lacking, or information is old and scarce. RESULTS: During this study, 8586 specimens belonging to 41 species were collected. We present the first faunistic data on Culicoides species in the former Yugoslav Republic of Macedonia (FYROM), Kosovo, Montenegro and Serbia. For other countries (Bosnia and Herzegovina, Bulgaria and Croatia), all historical records were compiled for the first time and then expanded with our findings to various extents. In all countries, confirmed or suspected BTV vector species belonging to the subgenera Avaritia and Culicoides were collected. The total number of species sampled during our field collections was 20 in Bosnia and Herzegovina (15 new records), 10 in Bulgaria (2 new records), 10 in Croatia (5 new records), 13 in FYROM, 9 in Kosovo, 15 in Montenegro, and 28 in Serbia. Of these, 14 species were registered for the first time in this part of the Balkans. CONCLUSIONS: This paper provides the first data about Culicoides fauna in FYROM, Kosovo, Montenegro and Serbia, as well as new records and an update on the checklists for Bosnia and Herzegovina, Bulgaria and Croatia. These findings provide preliminary insights into the routes of BTV introduction and spreading within the Balkans, and present a valuable contribution to further research related to Culicoides-borne diseases in Europe.

Imported malaria in Bulgaria, status and prognosis after eradication in 1965.

BACKGROUND: This retrospective analysis assessed all recorded malaria cases in Bulgaria after 1965, when the country was certified as malaria-free by the World Health Organization (WHO), and evaluated the readiness of the public health system to interrupt an outbreak of local transmission in case of infection importation. METHODS: The cases were analyzed according to causative species; geographic origin of the imported case; and the citizenship, age, and gender of the infected individuals. RESULTS: In the 50-year study period (1966-2015), there were a total of 3011 cases of malaria imported to Bulgaria from different regions of the world. The majority of the cases originating in Africa were caused by Plasmodium falciparum (65.5%), while most of these originating in Asia were caused by P. vivax (80.9%). The potential season for malaria transmission in Bulgaria is from April to October, and 58.5% of the malaria cases were imported during that time of the year. CONCLUSIONS: The increasing movement of people to and from areas endemic for malaria requires the health authorities of countries with appropriate conditions for reintroduction to conduct enhanced measures for surveillance and control of this potentially deadly disease.

Human Parasitic Diseases in Bulgaria in Between 2013-2014.

BACKGROUND: In Bulgaria, more than 20 autochthonous human parasitic infections have been described and some of them are widespread. Over 50 imported protozoan and helminthic infections represent diagnostic and therapeutic challenges and pose epidemiological risks due to the possibility of local transmission. AIMS: To establish the distribution of autochthonous and imported parasitic diseases among the population of the country over a 2-year period (2013-2014) and to evaluate their significance in the public health system. STUDY DESIGN: Cross sectional study. METHODS: We used the annual reports by regional health inspectorates and data from the National Reference Laboratory at the National Centre of Infectious and Parasitic Diseases on all individuals infected with parasitic diseases in the country. Prevalence was calculated for parasitic diseases with few or absent clinical manifestations (oligosymptomatic or asymptomatic infections). Incidence per 100.000 was calculated for diseases with an overt clinical picture or those that required hospitalisation and specialised medical interventions (e.g. surgery). RESULTS: During the research period, parasitological studies were conducted on 1441.244 persons, and parasitic infections were diagnosed in 22.039 individuals. Distribution of various parasitic pathogens among the population displayed statistically significant differences in prevalence for some intestinal parasites (enterobiasis 0.81%, giardiasis 0.34% and blastocystosis 0.22%). For certain zoonotic diseases such as cystic echinococcosis (average incidence of 3.99 per 100.000) and trichinellosis (average incidence of 0.8 per 100.000), the incidence exceeds several times the annual incidence recorded in the European Union. CONCLUSION: Parasitic diseases still pose a substantial problem with social and medical impacts on the residents of our country. Improved efficiency regarding autochthonous and imported parasitic diseases is essential in providing the public health system the tools it needs to combat these diseases. Attention should be focused on the various imported vector-borne parasitic diseases (e.g. malaria and cutaneous leishmaniasis) for which the country is potentially endemic.