Rapid increase in neuroinvasive West Nile virus infections in humans, Italy, July 2022.

As in 2018, when a large West Nile virus (WNV) epidemic occurred, the 2022 vector season in Italy was marked by an early onset of WNV circulation in mosquitoes and birds. Human infections were limited until early July, when we observed a rapid increase in the number of cases. We describe the epidemiology of human infections and animal and vector surveillance for WNV and compare the more consolidated data of June and July 2022 with the same period in 2018.

Non-imported malaria in Italy: paradigmatic approaches and public health implications following an unusual cluster of cases in 2017.

BACKGROUND: The European region achieved interruption of malaria transmission during the 1970s. Since then, malaria control programs were replaced by surveillance systems in order to prevent possible re-emergence of this disease. Sporadic cases of non-imported malaria were recorded in several European countries in the past decade and locally transmitted outbreaks of Plasmodium vivax, most probably supported by Anopheles sacharovi, have been repeatedly reported from Greece since 2009. The possibility of locally-transmitted malaria has been extensively studied in Italy where the former malaria vector An. labranchiae survived the control campaign which led to malaria elimination. In this study, we present paradigmatic cases that occurred during a 2017 unusual cluster, which caused strong concern in public opinion and were carefully investigated after the implementation of the updated malaria surveillance system. METHODS: For suspected locally-transmitted malaria cases, alerts to Ministry of Health (MoH) and the National Institute of Health (ISS) were mandated by the Local Health Services (LHS). Epidemiological investigations on the transmission modes and the identification of possible infection's source were carried out by LHS, MoH and ISS. Entomological investigations were implemented locally for all suspected locally-transmitted cases that occurred in periods suitable to anopheline activity. Molecular diagnosis by nested-PCR for the five human Plasmodium species was performed to support microscopic diagnosis. In addition, genotyping of P. falciparum isolate was carried out to investigate putative sources of infection and transmission modalities. RESULTS: In 2017, a cluster of seven non-imported cases was recorded from August through October. Among them, P. ovale curtisi was responsible of one case whereas six cases were caused by P. falciparum. Two cases were proved to be nosocomial while the other five were recorded as cryptic at the end of epidemiological investigations. CONCLUSIONS: The epidemiological evidence shows that the locally acquired events are sporadic, often remain unresolved and classified as cryptic ones despite investigative efforts. The "cluster" of seven non-imported cases that occurred in 2017 in different regions of Italy therefore represents a conscious alert that should lead us to maintain a constant level of surveillance in a former malaria endemic country.

Secondary Autochthonous Outbreak of Chikungunya, Southern Italy, 2017.

In 2017, a chikungunya outbreak in central Italy later evolved into a secondary cluster in southern Italy, providing evidence of disease emergence in new areas. Officials have taken action to raise awareness among clinicians and the general population, increase timely case detection, reduce mosquito breeding sites, and promote mosquito bite prevention.

Vector competence of Aedes albopictus for the Indian Ocean lineage (IOL) chikungunya viruses of the 2007 and 2017 outbreaks in Italy: a comparison between strains with and without the E1:A226V mutation.

We compared the vector competence of an Italian population of Aedes albopictus for two strains of chikungunya virus (CHIKV), with and without E1:A226V mutation, responsible for outbreaks in 2007 in the Emilia Romagna region and 2017 in the Lazio and Calabria regions, respectively. Ae. albopictus showed similar vector competence for both viral strains indicating that E1:A226V mutation is not exclusively responsible for ability of CHIKV to replicate well in this mosquito species.

Detection of a chikungunya outbreak in Central Italy, August to September 2017.

An autochthonous chikungunya outbreak is ongoing near Anzio, a coastal town in the province of Rome. The virus isolated from one patient and mosquitoes lacks the A226V mutation and belongs to an East Central South African strain. As of 20 September, 86 cases are laboratory-confirmed. The outbreak proximity to the capital, its late summer occurrence, and diagnostic delays, are favouring transmission. Vector control, enhanced surveillance and restricted blood donations are being implemented in affected areas.

Experimental studies of susceptibility of Italian Aedes albopictus to Zika virus.

We report a study on vector competence of an Italian population of Aedes albopictus for Zika virus (ZIKV). Ae. albopictus was susceptible to ZIKV infection (infection rate: 10%), and the virus could disseminate and was secreted in the mosquito's saliva (dissemination rate: 29%; transmission rate: 29%) after an extrinsic incubation period of 11 days. The observed vector competence was lower than that of an Ae. aegypti colony tested in parallel.

Experimental investigation of the susceptibility of Italian Culex pipiens mosquitoes to Zika virus infection.

We investigated the susceptibility of an Italian population of Culex pipiens mosquitoes to Zika virus (ZIKV) infection, tested in parallel with Aedes aegypti, as a positive control. We analysed mosquitoes at 0, 3, 7, 10, 14, 20 and 24 days after an infectious blood meal. Viral RNA was detected in the body of Cx. pipiens up to three days post-infection, but not at later time points. Our results indicate that Cx. pipiens is not susceptible to ZIKV infection.

Myiasis of the Tracheostomy Wound Caused by Sarcophaga (Liopygia) argyrostoma (Diptera: Sarcophagidae): Molecular Identification Based on the Mitochondrial Cytochrome c Oxidase I Gene.

Wound myiasis is the infestation of open wounds of mammalian hosts caused by larvae of various species of flies. This kind of myiasis can be a serious problem for immobilized patients with open wounds. Here, we identify a dipteran larva found in the tracheostomy wound of a child affected by a severe spinal muscular atrophy. The collected larva was dissected and microscopically analyzed. DNA was extracted from part of the larva and used for the molecular identification. A 487 bp fragment, including part of 5.8 S, the internal transcribed spacer 2 (ITS2), and part of 28S, was amplified using a novel PCR assay to be cloned and sequenced. The barcode region of cytochrome oxidase I (COI) was also cloned and sequenced after PCR amplification. The larva, designated as SASI1, was identified as a third instar of Sarcophaga sp. The COI sequencing confirmed a low similarity with Sarcophaga ruficornis (F.) (95%), yet COI showed a 100% similarity with Sarcophaga argyrostoma (Robineau-Desvoidy, 1830) species. Therefore, SASI1 was identified as a S. argyrostoma larva on the basis of its COI barcode. This is one of the rare cases of myiasis of tracheostomy wound and the first caused by S. argyrostoma.

Anopheles sacharovi in Italy: first record of the historical malaria vector after over 50 years.

BACKGROUND: Anopheles sacharovi, a member of the Anopheles maculipennis complex, was a historical malaria vector in Italy, no longer found since the last report at the end of 1960s. In September 2022, within the Surveillance Project for the residual anophelism, a single specimen of An. maculipennis sensu lato collected in Lecce municipality (Apulia region) was molecularly identified as An. sacharovi. This record led to implement a targeted entomological survey in September 2023. METHODS: Investigation was conducted in the areas around the first discovery, focusing on animal farms, riding stables and potential breeding sites. Adult and immature mosquitoes were collected, using active search or traps, in several natural and rural sites. Mosquitoes belonging to An. maculipennis complex were identified morphologically and molecularly by a home-made routine quantitative polymerase chain reaction (qPCR) assay, developed specifically for the rapid identification of An. labranchiae, and, when necessary, by amplification and sequencing of the ITS-2 molecular marker. RESULTS: Out of the 11 sites investigated, 6 were positive for Anopheles presence. All 20 An. maculipennis s.l. (7 adults, 10 larvae and 3 pupae) collected in the areas were identified as An. sacharovi by ITS-2 sequencing. CONCLUSIONS: The discovery of An. sacharovi, considered to have disappeared from Italy for over 50 years, has a strong health relevance and impact, highlighting an increase in the receptivity of the southern areas. As imported malaria cases in European countries are reported every year, the risk of Plasmodium introduction by gametocyte carriers among travellers from endemic countries should be taken into greater consideration. Our findings allow rethinking and building new models for the prediction and expansion of introduced malaria. Furthermore, to prevent the risk of reintroduction of the disease, the need to strengthen the surveillance of residual anophelism throughout the South should be considered.

Assessing the Risk of Dengue Virus Local Transmission: Study on Vector Competence of Italian Aedes albopictus.

The frequency of locally transmitted dengue virus (DENV) infections has increased in Europe in recent years, facilitated by the invasive mosquito species Aedes albopictus, which is well established in a large area of Europe. In Italy, the first indigenous dengue outbreak was reported in August 2020 with 11 locally acquired cases in the Veneto region (northeast Italy), caused by a DENV-1 viral strain closely related to a previously described strain circulating in Singapore and China. In this study, we evaluated the vector competence of two Italian populations of Ae. albopictus compared to an Ae. aegypti lab colony. We performed experimental infections using a DENV-1 strain that is phylogenetically close to the strain responsible for the 2020 Italian autochthonous outbreak. Our results showed that local Ae. albopictus is susceptible to infection and is able to transmit the virus, confirming the relevant risk of possible outbreaks starting from an imported case.

A modified BG-Sentinel trap equipped with FTA card as a novel tool for mosquito-borne disease surveillance: a field test for flavivirus detection.

Early detection of pathogens in vectors is important in preventing the spread of arboviral diseases, providing a timely indicator of pathogen circulation before outbreaks occur. However, entomological surveillance may face logistical constraints, such as maintaining the cold chain, and resource limitations, such as the field and laboratory workload of mosquito processing. We propose an FTA card-based trapping system that aims to simplify both field and laboratory phases of arbovirus surveillance. We modified a BG-Sentinel trap to include a mosquito collection chamber and a sugar feeding source through an FTA card soaked in a long-lasting viscous solution of honey and hydroxy-cellulose hydrogel. The FTA card ensures environmental preservation of nucleic acids, allowing continuous collection and feeding activity of specimens for several days and reducing the effort required for viral detection. We tested the trap prototype during two field seasons (2019 and 2021) in North-eastern Italy and compared it to CDC-CO2 trapping applied in West Nile and Usutu virus regional surveillance. Collections by the BG-FTA approach detected high species diversity, including Culex pipiens, Aedes albopictus, Culex modestus, Anopheles maculipennis sensu lato and Ochlerotatus caspius. When used for two-days sampling, the BG-FTA trap performed equally to CDC also for the WNV-major vector Cx. pipiens. The FTA cards detected both WNV and USUV, confirming the reliability of this novel approach to detect viral circulation in infectious mosquitoes. We recommend this surveillance approach as a particularly useful alternative in multi-target surveillance, for sampling in remote areas and in contexts characterized by high mosquito densities and diversity.

Molecular Characterization of Anopheles algeriensis Theobald, 1903 (Diptera: Culicidae) Populations from Europe.

Anopheles algeriensis Theobald, 1903, considered a competent vector of Plasmodium parasites, is a mosquito species widely distributed in the Mediterranean area but rare in Northern and Central Europe. The disappearance of its suitable breeding sites in Italy is having a detrimental effect on the occurrence of this species once common along the Southern coasts and on the islands. Recently, molecular investigations have renewed interest in this species, highlighting a genetic heterogeneity among European populations. In this study, An. algeriensis populations from Italy, Germany, Romania, and Sweden were analyzed by molecular typing of the intergenic transcribed spacer 2 (ITS2). The mitochondrial cytochrome c oxidase subunit I (COI) was also analyzed from specimens collected in Southern Italy. With the aim of investigating the population structure of this species, the obtained data were compared to all publicly available ITS2 and COI sequences of An. algeriensis, adding specimens from Spain and Portugal. The analyses of both markers indicate a split between Iberian populations (Spain for ITS2 and Spain/Portugal for COI) and those from the rest of Europe, revealing two cryptic species. The analysis of the COI barcode revealed a third clade representing a cryptic species present in Danube Delta (Romania). The high levels of genetic divergence among the clades of An. algeriensis indicate that this taxon represents a species complex, potentially harboring several distinct cryptic species.

A noteworthy record of Ornithodoros (Alectorobius) coniceps (Ixodida: Argasidae) from Central Italy.

The first record of Ornithodoros (Alectorobius) coniceps (Canestrini) was reported for Italy in 1877, inside the interstices of the ancient mosaics at S. Marco Basilica in Venice. Afterwards only few discoveries of the species are reported for Italy; the last record is dated back to 1984, in L'Aquila town (Abruzzo Region). The present study shows the data of a survey carried out as a result of a massive infestation by O. coniceps in an ancient villa in Anzio town (Latium region) recently restored. In the past decades the villa has been fallen into disrepair, becoming an occasional shelter for wild animals mainly pigeons, that colonized the whole building for generations. This case appears worthy of note because it is the first record of this species after more than 25 years in Italy. A total of 136 specimens were collected by three methods: manual, mechanical aspirators and Wilson traps. Wilson trapping indicates positive O. coniceps tropism for CO(2).

The common European mosquitoes Culex pipiens and Aedes albopictus are unable to transmit SARS-CoV-2 after a natural-mimicking challenge with infected blood.

BACKGROUND: On 11 March 2020, the World Health Organisation (WHO) declared the coronavirus disease 2019 (COVID-19) outbreak to be a pandemic. As the mosquito season progressed, the understandable concern that mosquitoes could transmit the virus began to increase among the general public and public health organisations. We have investigated the vector competence of Culex pipiens and Aedes albopictus, the two most common species of vector mosquitoes in Europe, for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Due to the very unusual feeding behaviour of Ae. albopictus, we also evaluated the role of this mosquito in a potential mechanical transmission of the virus. METHODS: For the vector competence study, mosquitoes were allowed to take several infectious blood meals. The mosquitoes were then collected and analysed at 0, 3, 7 and 10 days post-feeding. For the mechanical transmission test, Ae. albopictus females were allowed to feed for a short time on a feeder containing infectious blood and then on a feeder containing virus-free blood. Both mosquitoes and blood were tested for viral presence. RESULTS: Culex pipiens and Ae. albopictus were found not be competent vectors for SARS-CoV-2, and Ae. albopictus was unable to mechanically transmit the virus. CONCLUSIONS: This is the first study to show that the most common species of vector mosquitoes in Europe do not transmit SARS-CoV-2 and that Ae. albopictus is unable to mechanically transmit the virus from a positive host to a healthy host through host-feeding.

Monitoring Campaign over an Edible Dormouse Population (Glis glis; Rodentia: Gliridae) in Sicily: First Report of Mesocestodiasis.

This study reports on the health status of the edible dormouse (Glis glis) living in Nebrodi Park (Sicily, Italy), responsible for nut crop damage in the area. In the frame of a monitoring campaign for potential zoonotic risk involving 30 dormice, rectal and conjunctival swabs and fur and nest content were collected for bacteriological and parasitological examinations, respectively. A large presence of fleas belonging to Monopsyllus sciurorum was found. Necropsy of a dead dormouse revealed an infection of Mesocestoides lineatus, whose cysts were found in the abdomen cavity and on the liver; this is the first report of this in this species. Further studies are necessary to identify their role in the environment, considering the limited knowledge of this species in Italy.

Entomological Surveillance in Former Malaria-endemic Areas of Southern Italy.

Malaria still represents a potential public health issue in Italy, and the presence of former Anopheles vectors and cases imported annually merit continuous surveillance. In areas no longer endemic, the concurrent presence of gametocyte carriers and competent vectors makes re-emergence of local transmission possible, as recently reported in Greece. In October 2017, due to the occurrence of four suspected introduced malaria cases in the province of Taranto (Apulia region), entomological investigations were performed to verify the involvement of local anopheline species. In 2019-2020 entomological surveys were extended to other areas historically prone to malaria between the provinces of Taranto and Matera and the province of Foggia (Gargano Promontory). Resting mosquitoes were collected in animal shelters and human dwellings, larvae were sampled in natural and artificial breeding sites, and specimens were both morphologically and molecularly identified. A total of 2228 mosquitoes were collected, 54.3% of which were anophelines. In all the investigated areas, Anopheles labranchiae was the most widespread species, while Anopheles algeriensis was predominant at the Gargano sites, and Anopheles superpictus and Anopheles plumbeus were recorded in the province of Matera. Our findings showed a potentially high receptivity in the surveyed areas, where the abundance of the two former malaria vectors, An. labranchiae and An. superpictus, is related to environmental and climatic parameters and to anthropic activities.

An insight on drug resistance in Plasmodium vivax, a still neglected human malaria parasite.

Plasmodium vivax has been considered for years as responsible for a mild form of malaria, due to the absence in the majority of its infections of the severe form of the disease, typical instead of the deadly human parasite P. falciparum. In the last decade, studies on vivax malaria have had a partial step ahead especially after the completion of the whole genome project, but there is still a gap of knowledge in the biology of this parasite. Moreover, the emergence of P. vivax antimalarial resistance in 1980s and its subsequent spread in the Southeast Asia have indicated new concerns about the possibility to control this parasite. P. vivax drug resistance poses a major threat to endemic countries and without important international efforts, we could assist in a near future to the paradox of seeing different malaria co-endemic countries, that have successfully controlled/eliminated P. falciparum, still fighting against P. vivax.

MicroRNAs from saliva of anopheline mosquitoes mimic human endogenous miRNAs and may contribute to vector-host-pathogen interactions.

During blood feeding haematophagous arthropods inject into their hosts a cocktail of salivary proteins whose main role is to counteract host haemostasis, inflammation and immunity. However, animal body fluids are known to also carry miRNAs. To get insights into saliva and salivary gland miRNA repertoires of the African malaria vector Anopheles coluzzii we used small RNA-Seq and identified 214 miRNAs, including tissue-enriched, sex-biased and putative novel anopheline miRNAs. Noteworthy, miRNAs were asymmetrically distributed between saliva and salivary glands, suggesting that selected miRNAs may be preferentially directed toward mosquito saliva. The evolutionary conservation of a subset of saliva miRNAs in Anopheles and Aedes mosquitoes, and in the tick Ixodes ricinus, supports the idea of a non-random occurrence pointing to their possible physiological role in blood feeding by arthropods. Strikingly, eleven of the most abundant An. coluzzi saliva miRNAs mimicked human miRNAs. Prediction analysis and search for experimentally validated targets indicated that miRNAs from An. coluzzii saliva may act on host mRNAs involved in immune and inflammatory responses. Overall, this study raises the intriguing hypothesis that miRNAs injected into vertebrates with vector saliva may contribute to host manipulation with possible implication for vector-host interaction and pathogen transmission.

Analysis in a murine model points to IgG responses against the 34k2 salivary proteins from Aedes albopictus and Aedes aegypti as novel promising candidate markers of host exposure to Aedes mosquitoes.

BACKGROUND: Aedes mosquitoes are vectors of arboviral diseases of great relevance for public health. The recent outbreaks of dengue, Zika, chikungunya and the rapid worldwide spreading of Aedes albopictus emphasize the need for improvement of vector surveillance and control. Host antibody response to mosquito salivary antigens is emerging as a relevant additional tool to directly assess vector-host contact, monitor efficacy of control interventions and evaluate risk of arboviral transmission. METHODOLOGY/PRINCIPAL FINDINGS: Groups of four BALB/c mice were immunized by exposure to bites of either Aedes albopictus or Aedes aegypti. The 34k2 salivary proteins from Ae. albopictus (al34k2) and Ae. aegypti (ae34k2) were expressed in recombinant form and Ae. albopictus salivary peptides were designed through B-cell epitopes prediction software. IgG responses to salivary gland extracts, peptides, al34k2 and ae34k2 were measured in exposed mice. Both al34k2 and ae34k2, with some individual and antigen-specific variation, elicited a clearly detectable antibody response in immunized mice. Remarkably, the two orthologous proteins showed very low level of immune cross-reactivity, suggesting they may eventually be developed as species-specific markers of host exposure. The al34k2 immunogenicity and the limited immune cross-reactivity to ae34k2 were confirmed in a single human donor hyperimmune to Ae. albopictus saliva. CONCLUSIONS/SIGNIFICANCE: Our study shows that exposure to bites of Ae. albopictus or Ae. aegypti evokes in mice species-specific IgG responses to al34k2 or ae34k2, respectively. Deeper understanding of duration of antibody response and validation in natural conditions of human exposure to Aedes mosquitoes are certainly needed. However, our findings point to the al34k2 salivary protein as a promising potential candidate for the development of immunoassays to evaluate human exposure to Ae. albopictus. This would be a step forward in the establishment of a serological toolbox for the simultaneous assessment of human exposure to Aedes vectors and the pathogens they transmit.

Larvicidal activity of Ocimum campechianum, Ocotea quixos and Piper aduncum essential oils against Aedes aegypti.

Aedes aegypti, the main arbovirus vector of the Yellow fever, Dengue, Chikungunya and Zika viruses, is widely distributed in tropical and subtropical areas throughout the world. Preventive control efforts have been implemented worldwide aimed at reducing its impact on human health. The recent reduction of chemicals available for vector control due to their negative impact on the environment and human health and the increase in mosquito resistance to insecticides have driven the research community to identify and evaluate sustainable alternatives to synthetic insecticides. In this study, the potential larvicidal effect of essential oils extracted from Ocimum campechianum, Ocotea quixos and Piper aduncum were tested in vitro. GC and GC-MS analyses showed that the main compounds were eugenol (18%), 1,8-cineole (39%) and dillapiole (48%), respectively. Susceptibility to essential oils was measured according to the WHO protocol. After 24 h, the mean percentage mortality ranged from 2.7 to 100% for P. aduncum, from 2.2 to 100% for O. campechianum, and from 2.9 to 100% for O. quixos. The highest potential was displayed by P. aduncum, followed by O. campechianum and O. quixos, with LC50 values of 25.7, 69.3 and 75.5 ppm, respectively. The rapid and effective larvicidal activity of these three oils led us to consider these results to be promising, also considering the possibility of local cultivation of these plants in tropical and subtropical areas and the simple technology for their large-scale preparation and production. Further studies are needed to evaluate the individual components and their activity as larvicides.