DNA identification of species of the Anopheles maculipennis complex and first record of An. daciae in Belgium.

The present study aimed at identifying the members of the Anopheles maculipennis complex (Diptera: Culicidae) occurring in Belgium. Therefore, the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) and the mitochondrial cytochrome oxidase subunit I (COI) loci were sequenced in 175 and 111 specimens, respectively, collected between 2007 and 2019. In parallel, the suitability of two species-diagnostic PCR-RFLP assays was tested. The identified specimens included: An. maculipennis s.s. (N = 105), An. daciae (N = 62), An. atroparvus (N = 6) and An. messeae (N = 2). Each species was characterized by unique ITS2 haplotypes, whereas COI only supported the monophyly of An. atroparvus, a historical malaria vector in Belgium. Species identification results were further supported by unique PCR-RFLP banding patterns. We report for the first time An. daciae in Belgium, where it was found to co-occur with An. maculipennis s.s. The latter was the most prevalent in the collection studied (60%) and appears to have the widest distribution in Belgium. As in other studies, An. daciae and An. messeae appeared the most closely related species, up to the point that their species status remains debatable, while their ecological differences, including vector competences, need further study.

Targeting of blood safety measures to affected areas with ongoing local transmission of malaria.

An outbreak of locally acquired Plasmodium vivax malaria in Greece started in 2009 and peaked in 2011. Targeting of blood safety measures to affected areas with ongoing transmission of malaria raised questions of how to define spatial boundaries of such an area and when to trigger any specific blood safety measures, including whether and which blood donation screening strategy to apply. To provide scientific advice the European Centre for Disease Prevention and Control (ECDC) organised expert meetings in 2013. The outcomes of these consultations are expert opinions covering spatial targeting of blood safety measures to affected areas with ongoing local transmission of malaria and blood donation screening strategy for evidence of malaria infection in these areas. Opinions could help EU national blood safety authorities in developing a preventive strategy during malaria outbreaks.

The diagnostic accuracy of serological tests for Lyme borreliosis in Europe: a systematic review and meta-analysis.

BACKGROUND: Interpretation of serological assays in Lyme borreliosis requires an understanding of the clinical indications and the limitations of the currently available tests. We therefore systematically reviewed the accuracy of serological tests for the diagnosis of Lyme borreliosis in Europe. METHODS: We searched EMBASE en MEDLINE and contacted experts. Studies evaluating the diagnostic accuracy of serological assays for Lyme borreliosis in Europe were eligible. Study selection and data-extraction were done by two authors independently. We assessed study quality using the QUADAS-2 checklist. We used a hierarchical summary ROC meta-regression method for the meta-analyses. Potential sources of heterogeneity were test-type, commercial or in-house, Ig-type, antigen type and study quality. These were added as covariates to the model, to assess their effect on test accuracy. RESULTS: Seventy-eight studies evaluating an Enzyme-Linked ImmunoSorbent assay (ELISA) or an immunoblot assay against a reference standard of clinical criteria were included. None of the studies had low risk of bias for all QUADAS-2 domains. Sensitivity was highly heterogeneous, with summary estimates: erythema migrans 50% (95% CI 40% to 61%); neuroborreliosis 77% (95% CI 67% to 85%); acrodermatitis chronica atrophicans 97% (95% CI 94% to 99%); unspecified Lyme borreliosis 73% (95% CI 53% to 87%). Specificity was around 95% in studies with healthy controls, but around 80% in cross-sectional studies. Two-tiered algorithms or antibody indices did not outperform single test approaches. CONCLUSIONS: The observed heterogeneity and risk of bias complicate the extrapolation of our results to clinical practice. The usefulness of the serological tests for Lyme disease depends on the pre-test probability and subsequent predictive values in the setting where the tests are being used. Future diagnostic accuracy studies should be prospectively planned cross-sectional studies, done in settings where the test will be used in practice.

Sampling strategies for phlebotomine sand flies (Diptera: Psychodidae) in Europe.

The distribution of phlebotomine sand flies is widely reported to be changing in Europe. This can be attributed to either the discovery of sand flies in areas where they were previously overlooked (generally following an outbreak of leishmaniasis or other sand fly-related disease) or to true expansion of their range as a result of climatic or environmental changes. Routine surveillance for phlebotomines in Europe is localized, and often one of the challenges for entomologists working in non-leishmaniasis endemic countries is the lack of knowledge on how to conduct, plan and execute sampling for phlebotomines, or how to adapt on-going sampling strategies for other haematophagous diptera. This review brings together published and unpublished expert knowledge on sampling strategies for European phlebotomines of public health concern in order to provide practical advice on: how to conduct surveys; the collection and interpretation of field data; suitable techniques for the preservation of specimens obtained by different sampling methods; molecular techniques used for species identification; and the pathogens associated with sand flies and their detection methods.

An entomological review of invasive mosquitoes in Europe.

Among the invasive mosquitoes registered all over the world, Aedes species are particularly frequent and important. As several of them are potential vectors of disease, they present significant health concerns for 21st century Europe. Five species have established in mainland Europe, with two (Aedes albopictus and Aedes japonicus) becoming widespread and two (Ae. albopictus and Aedes aegypti) implicated in disease transmission to humans in Europe. The routes of importation and spread are often enigmatic, the ability to adapt to local environments and climates are rapid, and the biting nuisance and vector potential are both an ecomonic and public health concern. Europeans are used to cases of dengue and chikungunya in travellers returning from the tropics, but the threat to health and tourism in mainland Europe is substantive. Coupled to that are the emerging issues in the European overseas territorities and this paper is the first to consider the impacts in the remoter outposts of Europe. If entomologists and public health authorities are to address the spread of these mosquitoes and mitigate their health risks they must first be prepared to share information to better understand their biology and ecology, and share data on their distribution and control successes. This paper focusses in greater detail on the entomological and ecological aspects of these mosquitoes to assist with the risk assessment process, bringing together a large amount of information gathered through the ECDC VBORNET project.

Identification of Belgian mosquito species (Diptera: Culicidae) by DNA barcoding.

Since its introduction in 2003, DNA barcoding has proven to be a promising method for the identification of many taxa, including mosquitoes (Diptera: Culicidae). Many mosquito species are potential vectors of pathogens, and correct identification in all life stages is essential for effective mosquito monitoring and control. To use DNA barcoding for species identification, a reliable and comprehensive reference database of verified DNA sequences is required. Hence, DNA sequence diversity of mosquitoes in Belgium was assessed using a 658 bp fragment of the mitochondrial cytochrome oxidase I (COI) gene, and a reference data set was established. Most species appeared as well-supported clusters. Intraspecific Kimura 2-parameter (K2P) distances averaged 0.7%, and the maximum observed K2P distance was 6.2% for Aedes koreicus. A small overlap between intra- and interspecific K2P distances for congeneric sequences was observed. Overall, the identification success using best match and the best close match criteria were high, that is above 98%. No clear genetic division was found between the closely related species Aedes annulipes and Aedes cantans, which can be confused using morphological identification only. The members of the Anopheles maculipennis complex, that is Anopheles maculipennis s.s. and An. messeae, were weakly supported as monophyletic taxa. This study showed that DNA barcoding offers a reliable framework for mosquito species identification in Belgium except for some closely related species.

Public health significance of invasive mosquitoes in Europe.

There are currently five invasive Aedes mosquito species known to be established in Europe, namely Aedes albopictus, Aedes aegypti, Aedes japonicus, Aedes atropalpus and Aedes koreicus. Aedes albopictus and Aedes aegypti are the incriminated vectors in the recent outbreaks of chikungunya and dengue fever in Europe. However, both laboratory experiments and field observations indicate that these invasive mosquitoes have a potential to also transmit other pathogens of public health importance. Increasing travel and pathogen introduction, expansion of vector distribution, and both environmental and climatic changes are likely to raise the risk of pathogen transmission by these invasive Aedes mosquitoes. Their vector status and their involvement in pathogen transmission are dynamic processes that shape the future of mosquito-borne disease epidemiology in Europe. Beside vector surveillance, enhanced disease surveillance will enable the early detection of cases and the prompt implementation of control measures.

Mosquito-borne disease surveillance by the European Centre for Disease Prevention and Control.

For a few years, a series of traditionally tropical mosquito-borne diseases, such as chikungunya fever and dengue, have posed challenges to national public health authorities in the European region. Other diseases have re-emerged, e.g. malaria in Greece, or spread to other countries, e.g. West Nile fever. These diseases are reportable within the European Union (EU), and the European Centre for Disease Prevention and Control collects information in various ways to provide EU member states with topical assessments of disease threats, risks and trends for prompt and appropriate public health action. Using disease-specific expert networks, the European Surveillance System (TESSy) collects standardized comparable information on all statutory communicable diseases in a database. In addition, the event-based surveillance aims to detect potential public health threats early, and to allow timely response and support to blood deferral decisions for pathogens that can be transmitted through blood donation. Laboratory capacity for early detection is implemented through external quality assessments. Other activities include the development of guidelines for the surveillance of mosquito vectors, and the production of regularly updated maps on the currently known occurrence of mosquito vector species.

Nationwide inventory of mosquito biodiversity (Diptera: Culicidae) in Belgium, Europe.

To advance our restricted knowledge on mosquito biodiversity and distribution in Belgium, a national inventory started in 2007 (MODIRISK) based on a random selection of 936 collection points in three main environmental types: urban, rural and natural areas. Additionally, 64 sites were selected because of the risk of importing a vector or pathogen in these sites. Each site was sampled once between May and October 2007 and once in 2008 using Mosquito Magnet Liberty Plus traps. Diversity in pre-defined habitat types was calculated using three indices. The association between species and environmental types was assessed using a correspondence analysis. Twenty-three mosquito species belonging to traditionally recognized genera were found, including 21 indigenous and two exotic species. Highest species diversity (Simpson 0.765) and species richness (20 species) was observed in natural areas, although urban sites scored also well (Simpson 0.476, 16 species). Four clusters could be distinguished based on the correspondence analysis. The first one is related to human modified landscapes (such as urban, rural and industrial sites). A second is composed of species not associated with a specific habitat type, including the now widely distributed Anopheles plumbeus. A third group includes species commonly found in restored natural or bird migration areas, and a fourth cluster is composed of forest species. Outcomes of this study demonstrate the effectiveness of the designed sampling scheme and support the choice of the trap type. Obtained results of this first country-wide inventory of the Culicidae in Belgium may serve as a basis for risk assessment of emerging mosquito-borne diseases.

Bionomics of the established exotic mosquito species Aedes koreicus in Belgium, Europe.

Adults of an exotic mosquito, Aedes (Finlaya) koreicus (Edwards) (Diptera: Culicidae) were identified by morphology and genotyping from one site in Belgium in 2008. In late summer of that year, the occurrence of adults and immature stages reconfirmed its presence. This is the first record of this species outside its native range and in particular in Europe. Two subsites of the original location were prospected from April until October 2009 with different traps to evaluate the extent of its presence and establishment in the area and to understand the dynamics of the species' population. Next to Ae. koreicus, 15 other mosquito species were collected. Adult individuals of Ae. koreicus were found from May to September and larvae were still found early October. Larvae were mainly retrieved from artificial containers both in 2008 as in 2009. Containers with eggs and/or larvae were found up to 4 km away from the initial location, indicating the species is spreading locally. Though the introduction route is unknown, it may have occurred via international trade as a large industrial center was located nearby. A comparison of different climatic variables between locations in Belgium with Ae. koreicus and putative source locations in South Korea, revealed similarities between winter temperatures and the number of freezing days and nights in four consecutive years (2004-2008), while humidity and precipitation values differed strongly. The introduction of a new potential disease vector into Europe seems to be a result of proper entrance points, created by intense worldwide trade and suitable environmental conditions.

Autochthonous Plasmodium vivax malaria in Greece, 2011.

Between May and September 2011, twenty cases of Plasmodium vivax infection were reported in Greek citizens without reported travel history. The vast majority of those cases were confined to a delimited agricultural area of Evrotas, Lakonia. Conditions favouring locally acquired transmission of malaria, including the presence of competent vectors and migrants from endemic countries exist in Greece, underscoring the need for the development of an integrated preparedness and response plan for malaria prevention.

First two autochthonous dengue virus infections in metropolitan France, September 2010.

In September 2010, two cases of autochthonous dengue fever were diagnosed in metropolitan France for the first time. The cases occurring in Nice, southeast France, where Aedes albopictus is established, are evidence of dengue virus circulation in this area. This local transmission of dengue calls for further enhanced surveillance, active case finding and vector control measures to reduce the spread of the virus and the risk of an epidemic.

Personal protection by long-lasting insecticidal hammocks against the bites of forest malaria vectors.

OBJECTIVE: In Southeast Asia, malaria vectors bite outside the houses before bedtime, and forest dwellers rarely use insecticide-treated nets (ITNs). Thus, we tested the protection of long-lasting insecticidal hammocks (LLIH) using Olyset technology against exophagic vectors in two forest villages of Cambodia. METHODS: In each village, we conducted two entomological surveys (middle and end of the rainy season), each lasting 10 consecutive nights. These comprised human landing collections during the whole night by people sitting outside in the hammocks. Five households were selected per village, and for each household, two fixed positions were allocated: one for the control and one for the treated hammock. RESULTS: In total, 6449 mosquitoes were collected from control hammocks compared to 4481 in treated hammocks. Personal protection conferred by the hammocks was 46% (CI 95%: 35-55%) against the bites of Anopheles minimus. A significant reduction of An. dirus bites (46% CI 95%: 25-62%) was only observed at the end of the rainy season. An. maculatus and culicines bites were only reduced in one of the two study sites. CONCLUSION: Even if this LLIH is not inducing full protection against the bites of malaria vectors, it could prove effective in protecting forest workers and villagers before sleeping time. LLIH can be an additional and valuable tool in eliminating artemisinin-resistant malaria in the region.

Introduction and establishment of the exotic mosquito species Aedes japonicus japonicus (Diptera: Culicidae) in Belgium.

The establishment of the potential vector species Aedes (Finlaya) japonicusjaponicus (Theobald) (Diptera: Culicidae) in southern Belgium is reported. The species was most likely introduced through the international trade in used tires. It was first collected in 2002 on the premises of a second-hand tire company and was sampled using different sampling methods in the two consecutive years (2003-2004). It was only in 2007 and 2008, during a national mosquito survey (MODIRISK), that its presence as adults and larvae at the above-mentioned site and at another tire company in the area was confirmed based on morphological and molecular identification. This discovery is the first record for Belgium of an exotic mosquito species that established successfully and raises the question on the need for monitoring and control. Considering the accompanying species found during the surveys, we also report here the first observation of Culex (Maillotia) hortensis hortensis (Ficalbi) in Belgium.

Rhipicephalus appendiculatus and R. zambeziensis (Acari: Ixodidae) from Zambia: a molecular reassessment of their species status and identification.

The closely related species Rhipicephalus appendiculatus and R. zambeziensis co-occur in a wide area in Zambia. In this area, specimens of both species have been collected on the same individual host at the same time. In addition, specimens that are morphologically intermediate between R. appendiculatus and R. zambeziensis have been found in this area. These observations cast some doubt on the species status of R. appendiculatus and R. zambeziensis. Because the two taxa have varied influences on the epidemiology of East Coast fever (ECF), a cattle disease for which they act as vectors, it is essential that their taxonomic status is clarified and their identification is accurate. Therefore a phylogenetic analysis of nucleotide sequences of the ITS2 and a fragment of the mitochondrial 12S rDNA was performed to reassess the specific status of both taxa. This revealed two well supported clades coinciding with R. appendiculatus and R. zambeziensis as two separate species. In order to facilitate species identification a PCR-RFLP diagnostic assay was developed based on BauI digestion of the ITS2 gene. This assay produced clear diagnostic banding patterns for the two species and is applicable over a wide range of both species' distribution.

Review of the Minimus Complex of Anopheles, main malaria vector in Southeast Asia: from taxonomic issues to vector control strategies.

BACKGROUND: The Minimus Complex of Anopheles subgenus Cellia is composed of two sibling species, A and C, on the Southeast Asian mainland, and a third allopatric species E that occurs in the Ryukyu Archipelago (Japan), a malaria-free region. Anopheles minimus s.l. is considered to be one of the main malaria vector in the hilly forested regions of Southeast Asia. Despite a large number of studies over its range of distribution, it is difficult to have a global view of the ecological and bionomical characteristics of the individual species as different identification methods were used, generally without specific identification of the sibling species. OBJECTIVES: (1) To review the main malaria studies on An. minimus s.l.; (2) to discuss recently published data on the biology and ecology of each sibling species; and (3) to identify gaps in our understanding of the Minimus Complex. REVIEW RESULTS: Major biological and ecological trends are addressed, such as the high plasticity of trophic behaviour and the sympatry of species A and C over the Southeast Asian mainland. Despite the availability of rapid molecular identification methods, we still lack important information concerning the biological characteristics of each sibling species. These gaps must be filled in the future because An. minimus species A and C may exhibit different abilities to transmit malaria. CONCLUSION: We expect that entomological surveys will employ molecular methods to clearly identify these two species, and thus elucidate the biological characteristics of each species. As a consequence, current vector control strategies will be improved by targeting the most efficient vectors.

Restriction fragment length polymorphism method for the identification of major African and Asian malaria vectors within the Anopheles funestus and An. minimus groups.

The African Anopheles funestus and the Asian An. minimus groups are closely related and are probably considered distinct only because of their geographic separation. This study aimed at improving two identification methods based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) already developed for either group. Each PCR-RFLP, either on the internal transcribed spacer 2 (ITS2) for the An. minimus group, and domain 3 (D3) for the An. funestus group, was applied to the other group for the standardization of one identification method applicable on both continents. The ITS2 fragment digested by Bsi ZI showed the highest diagnostic power. This assay allowed the discrimination of at least 13 Anopheles species within the subgenus Cellia from two continents (Africa and Asia), among which are five major malaria vectors. Moreover, digestion of the D(3) with Msp I showed intragenomic variations within An. funestus populations. Two types of D3 copies (M and W) occurred in specimens from southern Africa. The populations from West-Central Africa presented only type W and East-Malagasy populations exhibited type M. Since An. funestus shows a great capacity of adaptation, these molecular variations, along with behavioral and ecologic ones, reinforce the hypothesis of a species complex that will need to be further investigated.