Outbreaks of autochthonous Dengue in Lazio region, Italy, August to September 2023: preliminary investigation.

Between August and September 2023, three distinct autochthonous dengue virus transmission events occurred in Lazio, Italy, with the main event in Rome. The events involved three different dengue serotypes. No link with previous imported cases was identified. Here we describe the epidemiological and phylogenetic analysis of the first autochthonous cases and the implemented control actions. The multiple transmission events call for a strengthening of the vector control strategies and future research to better characterise the risk in countries like Italy.

Microsatellite markers for Opisthorchis felineus to understand its genetic diversity and transmission patterns of opisthorchiosis.

Opisthorchis felineus is a food-borne trematode which causes opisthorchiosis and affects mainly the liver and bile ducts of the liver with a possible risk of bile duct carcinogenesis resulting in cholangiocarcinoma. In Russia, O. felineus is mainly endemic in Western Siberia (Ob and Irtysh river basins) and occurs throughout the Volga, Kama, Don, and Dnepr river basins. The prevalence, intensity, and clinical significance of human infections and the incidence of cholangiocarcinoma vary geographically in endemic regions. Currently, there is substantial evidence on genetic variation of O. felineus, but information on the population genetic structure is so far very scarce. Because microsatellite DNA of this parasite is not available, we for the first time isolated sufficient microsatellite loci to examine the genetic diversity and population structure of O. felineus, using multiple nuclear loci approach. A total of ten highly polymorphic microsatellite loci from a constructed enriched genomic DNA library were characterized, using 29 samples representing huge O. felineus metapopulation extended in latitude over 5000 km from Middle Europe to Western Siberia. At least three populations can be discerned as result of analysis of the microsatellite loci genetic diversity. Based on the results for the first time, a hypothesis was put forward about the formation of a modern habitat of O. felineus.

Detection of Endoparasites in Non-Native Raccoons from Central Italy.

The raccoon (Procyon lotor) is a carnivore native to North and Central America, gradually introduced into Asia and Europe, including Italy. It is an important carrier of multiple endoparasites, both Protozoa and Helminths, some of them being zoonotic. The aim of this study was to investigate the endoparasites of the non-native raccoon population of Central Italy. Sixty-two raccoons were collected by local competent authorities (sixty trapped and euthanized, two found dead) and subjected to necroscopic examination. Carcasses underwent a broad parasitological investigation, including coprological techniques (macroscopic examination of the gastrointestinal tract, lungs, trachea, and heart, Flotac®, Baermann test, and immunofluorescence for Giardia duodenalis and Cryptosporidium spp.), research on respiratory/urinary capillariosis and artificial digestion for Trichinella spp. larvae, and a histopathological examination of the ileum. Ascarid parasites were further identified at the species level using a next-generation sequencing-based amplicon sequencing approach. The results showed the presence of different Protozoa and Nematodes: Baylisascaris procyonis (26/62; 41.9%), Pearsonema sp. (6/62; 9.6%), Capillariidae (6/62; 9.6%), Eimeria sp. (2/62; 3.2%), Cryptosporidium sp. (2/62; 3.2%), and Ancylostomatidae (2/62; 3.2%). B. procyonis is an emerging helminthic zoonotic agent considered a serious concern for public and animal health, given the possibility of its transmission to paratenic hosts, including humans and pets. The demonstrated role of the raccoon as a multi-parasite carrier should be an incentive to continuing the eradication/control of this alien species, and supports the need to implement related disease surveillance programs.

Intestinal parasites infecting captive non-human primates in Italy.

Non-human primates (NHPs) living in captive conditions are susceptible to intestinal parasites that can contribute to mortality and morbidity, and cause zoonotic infections. Thus, parasite surveys on NHP populations under human care are relevant as part of the evaluation of NHPs welfare and in the zoonotic disease risk assessment, as well as in the exploration of parasite transmission pathways, according to the One-Health concept. This study aimed to identify intestinal parasites infecting NHPs living in two wildlife recovery centers and in a zoological garden, in Italy. Ninety-three fecal samples from Macaca tonkeana, Macaca fascicularis, Sapajus apella, Chlorocebus aethiops, Macaca fuscata, Macaca sylvanus, and Cebus capucinus were collected at Piano dell'Abatino Park (Lazio), and fecal smears and flotation were performed in order to identify parasites according to morphological keys. Additionally, one carcass of M. fuscata from the Bioparco Zoological Garden of Rome (Lazio) and one of M. fascicularis from the Center for the Recovery of Exotic and Maremma Wild Animals (Tuscany) were necropsied and intestinal adult nematodes were collected and characterized at morphological and molecular level, using the mitochondrial cox1 and rrnL markers. Protozoans (Entamoeba coli, Iodamoeba bütschlii, Dientamoeba fragilis-like, Giardia sp.), chromists (Balantidium/Buxtonella sp.) and nematodes (Capillaria sp., Trichuris sp., strongyliform larvae and Oesophagostomum sp.) were found through fecal smears and flotation. The collected adult nematodes from dead NHPs were morphologically identified as whipworms (genus Trichuris). Phylogenetic analyses grouped Trichuris specimens into the Trichuris trichiura complex of species, with specimens from M. fuscata clustering into a host-specific branch, and whipworms from M. fascicularis clustering within a clade formed by Trichuris infecting several primate species, including humans. The results here collected revealed the presence of potentially zoonotic parasites circulating in captive primates in Italy, providing useful information for the formulation of management and care plans for captive NHPs, and for the elaboration of safety measures for visitors and animal keepers.

Is Asian tiger mosquito (Aedes albopictus) going to become homodynamic in Southern Europe in the next decades due to climate change?

The Asian tiger mosquito, Aedes albopictus, competent vector of several arboviruses, poses significant impact on human health worldwide. Although global warming is a driver of A . albopictus range expansion, few studies focused on its effects on homodynamicity (i.e. the ability to breed all-year-round), a key factor of vectorial capacity and a primary condition for an Aedes-borne disease to become endemic in temperate areas. Data from a 4-year monitoring network set in Central Italy and records from weather stations were used to assess winter adult activity and weekly minimum temperatures. Winter oviposition occurred in 38 localities with a seasonal mean photoperiod of 9.7 : 14.3 (L : D) h. Positive collections (87) occurred with an average minimum temperature of the two and three weeks before sampling of approximately 4°C. According to these evidences and considering the climate projections of three global climate models and three shared socio-economic pathways for the next three 20-year periods (from 2021 to 2080), the minimum temperature of January will increase enough to allow an all-year-round oviposition of A . albopictus in several areas of the Mediterranean Basin. Due to vector homodynamicity, Aedes-borne diseases could become endemic in Southern Europe by the end of the twenty-first century, worsening the burden on human health.

Detection of viruses from feces of wild endangered Macaca maura: a potential threat to moor macaque survival and for zoonotic infection.

BACKGROUND: To date, there is a scarcity of information and literature on Macaca maura health status relative to viral diseases. The objectives of the present study were to investigate on the potential spread of enteric and non-enteric viruses shed in the environment through a wild macaque feces and to understand the possible interrelation in the spread of zoonotic viruses in a poorly studied geographical area, the Sulawesi Island. This study will also contribute providing useful information on potential threats to the health of this endangered species. METHODS: The sampling was conducted between 2014 and 2016 in the Bantimurung Bulusaraung National Park, in the south of the Sulawesi Island and non-invasive sampling methods were used to collect fresh stools of the M. maura, one of the seven macaque species endemic to the island of Sulawesi, Indonesia. The population under study consisted in two wild, neighboring social macaque groups with partially overlapping home ranges; twenty-four samples were collected and examined using negative staining electron microscopy and a panel of PCR protocols for the detection of ten RNA and two DNA viruses. RESULTS: Viral particles resembling parvovirus (5 samples), picornavirus (13 samples) and calicivirus (13 samples) were detected by electron microscopy whereas the PCR panel was negative for the 12 viruses investigated, except for one sample positive for a mosquito flavivirus. The results did not correlate with animal sex; furthermore, because all of the animals were clinically healthy, it was not possible to correlate feces consistency with viral presence. CONCLUSIONS: As information on viral infections in wild moor macaques remains limited, further studies are yet required to identify the fecal-oral and blood transmitted potentially zoonotic viruses, which may infect the moor macaque and other macaque species endemic to the South Sulawesi Island.

Blackflies (Simulium spp.) attacks on humans and animals in Rome and surrounding areas (Central Italy).

BACKGROUND: Due to their abundance in some environments, the blood-sucking habit and the involvement in the transmission of several diseases, blackflies of the genus Simulium (Diptera: Simuliidae) can be considered among the most annoying biting arthropods. METHODS: Following repeated attacks to humans and animals, entomological investigations were carried out in green areas of Rome and surroundings. Site and period of attacks were reported, together with the human and animal reactions to the bites. RESULTS: Four Simulium species have been identified through morphological or molecular analysis: Simulium intermedium, Simulium lineatum, Simulium lundstromi and Simulium ornatum (complex). Larval breeding sites were identified in small moats, where a high density of blackflies larvae and pupae was revealed. CONCLUSIONS: Being able to thrive in highly polluted water, even in few mm depth watercourses, some blackfly species are widely distributed in the area and their bites caused mild to severe reactions to humans and animals.

Systemic Infection by Angiostrongylus vasorum in a Fennec (Vulpeszerda) in an Italian Zoological Garden.

This paper reported a case of a metastrongyloid nematode Angiostrongylus vasorum infection in a fennec (Vulpes zerda) kept in a zoo in central Italy. The fennec had shown paralysis of the hind limbs, anorexia, weakness and respiratory signs before death. Cardiomegaly and granulomatous pneumonia were the major anatomopathological findings. Inflammatory lesions associated with parasitic larvae were observed in the lungs, brain, liver, heart, spinal cord and kidney of the fennec at histology. A. vasorum diagnosis was confirmed by both morphological and molecular identification of adult worms recovered at necropsy. Fennecs are active predators and maintain their hunting behaviour in captivity. Hence, it is likely that the animal was exposed to infection by preying on parasitised gastropods, intermediate hosts of A. vasorum, entering zoo enclosures from the surrounding environment. This is the first report of A. vasorum systemic infection in a captive fennec (V. zerda) in a zoo in Italy.

Delusional parasitosis: an entomological perspective after a 20-years-experience in two public medical and veterinary entomology laboratories.

Delusional parasitosis (DP) is psychiatric disorder characterized by the unshakeable belief of being infested by endo- or ectoparasites, without any evidence of infestations. Hence, DP sufferers often consult medical entomologists or dermatologists, rather than seeking help from a mental health practitioner. Here we present 39 cases of suspected DP occurred in twenty years in two Italian public medical entomology laboratories, to highlight their common features and peculiarities, based on the interviews and material brought to our laboratories. Our results confirmed that DP affects both sexes, but is more frequent in females over the age of 50 years. Cases of psychological contagion, mostly affecting elderly people, were reported within seven family units. Some rare or unique features of DP sufferers involved: the fixed belief to be infested by endoparasites, olfactory hallucinations of unpleasant smell, self-harm to get rid of the imaginary parasite and suicidal thoughts. We suggest the term "Digital pics sign" to describe a modern derivative of the "Matchbox sign", where the DP sufferers showed pictures and videos, in person or sent by email, as proofs of being infested. Medical entomologists, being the most frequently contacted professionals by DP sufferers, may be crucial to raise awareness about this psychological disorder and to encourage a multidisciplinary approach amongst medical professionals and pest control operators.

Strongyloides stercoralis in a dog litter: Evidence suggesting a transmammary transmission.

Strongyloides stercoralis is a soil-transmitted helminth with an unusual life cycle, causing canine and human strongyloidiasis, mainly endemic in tropical and subtropical areas. Following percutaneous or oral transmission of infective third-stage larvae in the vertebrate host, the parasite can cause autoinfection, leading to life-long infection. At present, the transmammary transmission was only assessed in experimentally infested dogs. Here, we provide observational evidence of S. stercoralis transmammary transmission in puppies suckling from a truffle dog from Central Italy, from where its presence was neglected.

Aedes albopictus abundance and phenology along an altitudinal gradient in Lazio region (central Italy).

BACKGROUND: The Asian tiger mosquito Aedes albopictus (Skuse 1894), which is native to Southeast Asia, is among the top 100 invasive species worldwide and one of the most troubling vector species. It has become established in more than 20 European countries. Since its arrival in Italy in the 1990s, the species has colonized all the regions of the country, up to an altitude of 600 m. Nevertheless, no thorough investigation has ever been performed to confirm or extend its elevation limit (EL) in Italy. METHODS: To define the EL of Ae. albopictus and analyse its phenology along an altitudinal gradient, we carried out an investigation by means of ovitraps placed in Lazio region, central Italy. Sampling was performed on a weekly basis in 13 villages within five 200-m altitudinal ranges [0-1000 m above sea level (asl)], with the addition of higher localities to the species range whenever the species was recorded in the highest range. RESULTS: Aedes albopictus has colonized sites well beyond its known EL, with established populations at 900 m asl and positive ovitraps recorded at 1193 m asl. The relationship between egg abundance and elevation was described by an exponential decay regression, which predicted an EL for oviposition at 1015 m asl. In the active season, egg-laying started earlier at low altitude and ended earlier within the highest altitudinal range. Aedes albopictus abundance and activity period (number of days active) decreased, respectively, by 95% and 34% from the lowest to the highest altitudinal range. CONCLUSIONS: Using data from the present study, the altitudinal limit of Ae. albopictus in central Italy was updated from 600 to 900 m asl. In addition, established populations were predicted to exist up to 1015 m asl. Considering that up to 99.5% of Lazio region's inhabitants could potentially be affected by Aedes-borne virus outbreaks, the surveillance area for Ae. albopictus should be expanded accordingly. However, our results also indicate that Ae. albopictus surveillance programs need to be revised in order to harmonize the resources earmarked for these with the altitudinal changes in the phenology of this species.

Giardia duodenalis in Wildlife: Exploring Genotype Diversity in Italy and across Europe.

Fragmented data are so far available on genotype diversity of G. duodenalis in wildlife in different countries in Europe, in particular, in Italy. In the present study, G. duodenalis sequences obtained from different Italian wild animals [12 porcupines (Hystrix cristata), 4 wild boars (Sus scrofa), 1 wolf (Canis lupus italicus), 6 Alpine chamois (Rupicapra rupicapra rupicapra)] were compared with those available from wild host species in Europe to add new data on the geographic distribution of Giardia assemblages/sub-assemblages and their transmission patterns among natural hosts. Thirty-eight sequences were obtained by MLG analysis (SSU-rRNA, bg, gdh, and tpi genes) and subsequently compared by phylogenetic and network analyses with those from wild species monitored in the last decades in Europe. The results revealed the presence of potentially zoonotic (A-AI, A-AII from wild boar; B from porcupine) and host-adapted (D from wolf; E, A-AIII from chamois) assemblages and sub-assemblages and represent the first report for Italian wild boar. The analysis did not find any evidence of spatial or host segregation for specific genetic variants, mostly shared between different hosts from different European countries. However, conflicting evidence was found in genotypic assignment, advocating for data improvement and new genomic approaches.

First report of the zoonotic nematode Baylisascaris procyonis in non-native raccoons (Procyon lotor) from Italy.

Baylisascaris procyonis is a nematode parasite of the raccoon (Procyon lotor), and it can be responsible for a severe form of larva migrans in humans. This parasite has been reported from many countries all over the world, after translocation of its natural host outside its native geographic range, North America. In the period between January and August 2021, 21 raccoons were cage-trapped and euthanized in Tuscany (Central Italy), in the context of a plan aimed at eradicating a reproductive population of this non-native species. All the animals were submitted for necroscopic examination. Adult ascariids were found in the small intestine of seven raccoons (prevalence 33.3%). Parasites have been identified as B. procyonis based on both morphometric and molecular approaches. The aim of the present article is to report the first finding of this zoonotic parasite from Italy, highlighting the sanitary risks linked to the introduction of alien vertebrate species in new areas.

Giardia duodenalis in colony stray cats from Italy.

Giardia duodenalis is the most common intestinal protozoan in humans and animals worldwide, including eight morphologically identical assemblages, infecting pets, livestock, wildlife and human beings. Assemblages A and B are those with the higher zoonotic potential, and they have been detected in several mammals other than humans; the others (C to H) show a higher host specificity. Cats can harbour both the specific Assemblage F and the zoonotic ones A and B. Several studies have been carried out on G. duodenalis genotypes in cats; however, the role of this species in the epidemiology of giardiasis is still poorly understood. In this scenario, the present study carried out the detection and genetic characterization at sub-assemblage level of G. duodenalis from colony stray cats in central Italy. In the period 2018-2019, 133 cat faecal samples were analysed for the presence of G. duodenalis cysts by a direct immunofluorescence assay. Positive samples were subsequently subjected to molecular analyses for assemblage/sub-assemblage identification. Forty-seven samples (35.3%) were positive for G. duodenalis cysts by immunofluorescence. G. duodenalis DNA was amplified at SSU-rDNA locus from 39 isolates: 37 were positive for zoonotic Assemblage A and 2 showed a mixed infection (A + B). Positive results for the ß-giardin gene were achieved for 25 isolates. Sequence analysis revealed 16 isolates belonging to Sub-assemblage AII and 8 to Sub-assemblage AIII. One isolate resulted as ambiguous AI/AIII. Large sequence variability at the sub-assemblage level was detected, with several double peaks and mutations, making complex a proper isolate allocation. When compared with previous studies, the 35.3% prevalence of G. duodenalis in cats reported in the present article was surprisingly high. Moreover, all positive cats resulted to be infected with zoonotic assemblages/sub-assemblages, thus indicating stray cats as a possible source of human giardiasis and highlighting the sanitary relevance of cat colonies in the study area.

First Report of Culicoides Biting Midges (Diptera: Ceratopogonidae) Attacking People in Italy, With the Description of Extreme Larval Breeding Sites and Diurnal Activity of Culicoides riethi.

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) play a paramount role in medical and veterinary entomology worldwide, particularly as vectors of pathogens which cause animal diseases. Biting midges are also infamous for the nuisance they provoke to people involved in outdoor activities. Nonetheless, attacks to man by midges from any Culicoides species have not been reported in Italy. An entomological investigation was performed following repeated attacks to man in a nature park near Rome (central Italy). The study area is a natural degassing zone, characterized by widespread hazardous gas emissions of CO2 and H2S, with several water bodies including permanent lakes, ponds, and pools. The biting midge C. riethi Kieffer, 1914 was very active during daytime in the period April-June. The species has been identified as responsible for attacks on people in the area. An in-depth analysis of the extreme environmental conditions revealed the ability of larvae to thrive in several water bodies, characterized by an extremely low pH and a high concentration of sulfates.

Plasmodium matutinum Causing Avian Malaria in Lovebirds (Agapornis roseicollis) Hosted in an Italian Zoo.

Avian malaria is a worldwide distributed, vector-born disease of birds caused by parasites of the order Haemosporida. There is a lack of knowledge about the presence and pathogenetic role of Haemosporida in Psittacidae. Here we report a case of avian malaria infection in lovebirds (Agapornis roseicollis), with the genetic characterization of the Plasmodium species involved. The birds were hosted in a zoo located in Italy, where avian malaria cases in African penguins (Spheniscus demersus) were already reported. Animals (n = 11) were submitted for necropsy after sudden death and were subjected to further analyses including histopathology, bacteriology, and PCR for the research of haemosporidians. Clinical history, gross lesions and histopathological observation of schizonts, together with positive PCR results for Plasmodium spp., demonstrated that avian malaria was the cause of death for one animal and the possible cause of death for the other nine. The sequences obtained were compared using BLAST and analyzed for similarity to sequences available at the MalAvi database. Genetic analyses demonstrated a 100% nucleotide identity to Plasmodium matutinum LINN1 for all the obtained sequences. To our knowledge, this is the first report describing avian malaria in lovebirds.

West Nile and Usutu viruses co-circulation in central Italy: outcomes of the 2018 integrated surveillance.

BACKGROUND: West Nile (WNV) and Usutu (USUV) are emerging vector-borne zoonotic flaviviruses. They are antigenically very similar, sharing the same life cycle with birds as amplification host, Culicidae as vector, and man/horse as dead-end host. They can co-circulate in an overlapping geographic range. In Europe, surveillance plans annually detect several outbreaks. METHODS: In Italy, a WNV/USUV surveillance plan is in place through passive and active surveillance. After a 2018 WNV outbreak, a reinforced integrated risk-based surveillance was performed in four municipalities through clinical and serological surveillance in horses, Culicidae catches, and testing on human blood-based products for transfusion. RESULTS: Eight WNV cases in eight equine holdings were detected. Twenty-three mosquitoe catches were performed and 2367 specimens of Culex pipiens caught; 17 pools were USUV positive. A total of 8889 human blood donations were tested, and two asymptomatic donors were USUV positive. CONCLUSIONS: Different surveillance components simultaneously detected WNV only in horses and USUV only in humans and mosquitoes. While in endemic areas (i.e. northern Italy) entomological surveillance is successfully used as an early detection warning, this method in central Italy seems ineffective. To achieve a high level of sensitivity, the entomological trapping effort should probably exceed a reasonable balance between cost and performance. Besides, WNV/USUV early detection can be addressed by horses and birds. Further research is needed to adapt the surveillance components in different epidemiological contexts.

Plasmodium matutinum Transmitted by Culex pipiens as a Cause of Avian Malaria in Captive African Penguins (Spheniscus demersus) in Italy.

Avian malaria is a parasitic disease of birds caused by protozoa belonging to the genus Plasmodium, within the order Haemosporida. Penguins are considered particularly susceptible, and outbreaks in captive populations can lead to high mortality. We used a multidisciplinary approach to investigate the death due to avian malaria, occurred between 2015 and 2019, in eight African penguins (Spheniscus demersus) kept in two Italian zoos located in central Italy, and situated about 30 km apart. We also provided information about the presence and circulation of Plasmodium spp. in mosquitoes in central Italy by sampling mosquitoes in both zoos where penguin mortalities occurred. In the eight dead penguins, gross and histopathological lesions were consistent with those previously observed by other authors in avian malaria outbreaks. Organs from dead penguins and mosquitoes collected in both zoos were tested for avian malaria parasites by using a PCR assay targeting the partial mitochondrial conserved region of the cytochrome b gene. Identification at species level was performed by sequencing analysis. Plasmodium matutinum was detected in both dead penguins and in mosquitoes (Culex pipiens), while Plasmodium vaughani in Culex pipiens only. Parasites were not found in any of the PCR tested Aedes albopictus samples. Based on our phylogenetic analysis, we detected three previously characterized lineages: Plasmodium matutinum LINN1 and AFTRU5, P. vaughani SYAT05. In Culex pipiens we also identified two novel lineages, CXPIP32 (inferred morphospecies Plasmodium matutinum) and CXPIP33 (inferred morphospecies P. vaughani). Significantly, LINN1 and AFTRU5 were found to be associated to penguin deaths, although only LINN1 was detected both in penguins (along the years of the study) and in Culex pipiens, while AFTRU5 was detected in a single penguin dead in 2017. In conclusion, in our study Plasmodium matutinum was found to cause avian malaria in captive penguins kept in Europe, with Culex pipiens being its most probable vector. Our results are in agreement with previous studies suggesting that Culex pipiens is one of the main vectors of Plasmodium spp. in Europe and the Northern Hemisphere. Zoos maintaining captive penguins in temperate areas where Culex pipiens is abundant should be well aware of the risks of avian malaria, and should put every effort to prevent outbreaks, in particular during the periods when the number of vectors is higher.

First evidence of blaNDM-1 and blaOXA-23 carbapenemase genes in human body lice infesting a second-hand T-shirt in a street market in Italy.

BACKGROUND: The spread of carbapenems resistance is a public health concern. The main group of carbapenemases encoding the ß-lactamases activity (bla genes) is the Metallo-ß-lactamases (MBLs). METHODS: The presence of carbapenemase blaOXA-23-like, blaOXA-40-like, blaOXA-51-like, blaOXA-58-like, and blaNDM-1 genes was screened by real time PCR in 26 Pediculus humanus insects identified from second-hand clothes in a local market in Central Italy. Bacteria diversity was also characterized through shotgun metagenomic amplification for a deep sequencing of the host-associated bacterial microbiomes. RESULTS: The blaOXA-23 and blaNDM-1 carbapenemases genes were found and metagenomic analysis showed a great presence of Acinetobacter species. CONCLUSIONS: These results suggest a new potential transmission path for carbapenemase gene spread through bacteria ingested by insects infesting humans.

A Novel Allele Specific Polymerase Chain Reaction (AS-PCR) Assay to Detect the V1016G Knockdown Resistance Mutation Confirms Its Widespread Presence in Aedes albopictus Populations from Italy.

Polymerase chain reaction (PCR)-based genotyping of mutations in the voltage-sensitive sodium channel (vssc) associated with resistance to pyrethroid insecticides is widely used and represents a potential early warning and monitoring system for insecticide resistance arising in mosquito populations, which are vectors of different human pathogens. In the secondary vector Aedes albopictus-an Asian species that has invaded and colonized the whole world, including temperate regions-sequencing of domain II of the vssc gene is still needed to detect the V1016G mutation associated with pyrethroid resistance. In this study we developed and tested a novel allele-specific PCR (AS-PCR) assay to genotype the V1016G mutation in this species and applied it to the analysis of wild populations from Italy. The results confirm the high accuracy of the novel AS-PCR and highlight frequencies of the V1016G allele as >5% in most sampling sites, with peaks of 20-45% in coastal touristic sites where pyrethroid treatments are extensively implemented, mostly for mosquito nuisance reduction. The high frequency of this mutation observed in Italian Ae. albopictus populations should serve as a warning bell, advocating for increased monitoring and management of a phenomenon which risks neutralizing the only weapon today available to counteract (risks of) arbovirus outbreaks.