BACKGROUND:Artemisinin-based combination therapy for Plasmodium falciparum malaria is threatened by the emergence of partial resistance. METHODS:Molecular characterization of antimalarial resistance-associated genes Pfk13, Pfcrt, Pfmdr1, Pfdhfr, Pfdhps and PfCytB was performed on blood samples collected from two patients with recurrent episodes of P. falciparum malaria. RESULTS:Two severe P. falciparum malaria in Italian travelers from sub-Saharan Africa showed recrudescence after standard treatment with intravenous artesunate and oral dihydroartemisinin-piperaquine. In one case, the validated marker R561H of artemisinin partial resistance was identified. In the second patient, no Pfkelch13 mutations were detected, and the recrudescent episode was probably the consequence of low blood drug concentration administered by nasogastric tube, although the possible role of Pfkelch13-independent pathways or reduced artemisinin susceptibility cannot be ruled out. CONCLUSION:The first imported case of artemisinin-resistant P. falciparum in Italy from sub-Saharan Africa underscores the need for continuous surveillance and eventually adapting treatment protocols.

We report 3 cases of babesiosis in Italy caused by Babesia species that are rarely reported in humans. The circulation of Babesia spp. among vectors, animals, and humans might be more common than previously thought, and babesiosis might be an underdiagnosed and emerging disease in Italy and Europe.

Anopheles superpictus (subgenus Cellia) plays an efficient role in malaria transmission in countries of the Mediterranean basin, Eastern Europe, and the Caucasus region, where it has been involved in the transmission of both Plasmodium falciparum and P. vivax. In Italy, this species was historically considered a secondary malaria vector, primarily recorded in the South including Sicily, along small rivers. It was less frequently observed in central Italy, only rarely reported in the North. Between 2022 and 2024, as part of routine investigations on residual anophelism, An. superpictus specimens were collected for the first time in the southern Maremma Plain (Magliano in Toscana, Grosseto, Tuscany). Adult mosquitoes were collected in animal shelters and tool premises of two farms using traps and manual aspiration methods. Larval sampling was performed by exploring potential breeding sites along the Albegna riverbed. Anophelinae adults and larvae were morphologically identified at the species level, and subsamples were molecularly confirmed by sequencing the cytochrome c oxidase subunit 1 gene and the ribosomal DNA internal transcribed spacer 2 region. A total of 1,106 adult mosquitoes were collected from July to October 2022. Anopheles superpictus was the most abundant species recorded (n = 512; 46

BackgroundMalaria is primarily transmitted through mosquito bites; occasionally, direct transmission through blood has been reported. Healthcare-associated infections refer to infections acquired in a hospital or another healthcare setting.AimThis systematic review aims to explore determinants of healthcare-associated malaria (HAM) cases.MethodThis review follows the PRISMA guidelines and was registered in PROSPERO (CRD42022309701). We searched five databases for publications on HAM cases published between 1 January 2000 and 7 December 2023. We initiated a data call for HAM cases to public health authorities from 37 European countries. We performed a backward and forward search, reviewed health authorities' websites, performed searches on Google and the European Scientific Conference on Applied Infectious Disease Epidemiology (ESCAIDE) conference abstracts book.ResultsWe identified 37 studies on HAM comprising 55 HAM cases, of which 35 (64%) were infected in Europe, primarily in Spain (nine cases), France and Italy (seven cases each). All cases were infected with Plasmodium falciparum except one individual. Fifty HAM cases were hospital inpatients and five were healthcare workers. Five patients died. Flushing of vascular catheters with contaminated heparin/saline solution and manipulation of intravenous catheters were the most frequently reported procedures leading to infection among patients.ConclusionsWhile rare, HAM transmission can be fatal. Healthcare-associated malaria is preventable through strict adherence to infection prevention and control procedures. Despite extensive investigations, the procedure leading to infection often remained unknown, highlighting the complexity of investigations. Guidance and protocols for conducting investigations may improve the success rate of such inquiries.

The oocyst is a sporogonic stage of Plasmodium development that takes place in the mosquito midgut in about 2 weeks. The cyst is protected by a capsule of unknown composition, and little is known about oocyst biology. We carried out a proteomic analysis of oocyst samples isolated at early, mid, and late time points of development. Four biological replicates for each time point were analyzed, and almost 600 oocyst-specific candidates were identified. The analysis revealed that, in young oocysts, there is a strong activity of protein and DNA synthesis, whereas in mature oocysts, proteins involved in oocyst and sporozoite development, gliding motility, and invasion are mostly abundant. Among the proteins identified at early stages, 17 candidates are specific to young oocysts. Thirty-four candidates are common to oocyst and the merosome stages (sporozoite proteins excluded), sharing common features as replication and egress. Western blot and immunofluorescence analyses of selected candidates confirm the expression profile obtained by proteomic analysis.

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.

Abstract 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. Graphical Abstract

BACKGROUND:In Italy, surveillance through mandatory notification of cases to the National Surveillance System (NSS) has shown evidence of underreporting over the years. To evaluate the overall quality of malaria dataset, Hospital Discharge Records (HDRs) were analyzed as a second data source.METHODS:Malaria cases by NSS and by HRDs were compared and analyzed from 2011-through 2017. The impact of cases was estimated by annual rates per 100,000 residents.RESULTS:Cases reported to NSS and to HDRs were 5,149 and 6,446, respectively. The annual rate recorded by NSS increased from 1.2 per 100,000 in 2011 to 1.4 per 100,000 in 2017, a similar trend was shown by HDRs, from 1.4 per 100,000 in 2011 to 1.6 per 100,000 in 2017. Every year, the number of NSS cases was lower than HDRs cases suggesting moderate underreporting of the mandatory notification. In both data sources adult males aged 25 to 44, and non-Italian travellers visiting friends and relatives were the most affected groups; Plasmodium falciparum was the prevalent agent identified, being the imported cases originated mainly from sub-Saharan Africa. As places of diagnosis and care, both data sources indicated hospitals located in Northern Italy in over 70% of cases.CONCLUSIONS:Although the comparison of malaria cases highlighted some underreporting by NSS, a fair agreement between the two institutional information systems was observed. The use of both data sources improves the performance of malaria surveillance in Italy, essentially for early warning systems in case of locally-acquired events and primary prevention in international travellers.

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.

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.

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.

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.

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.

Both malaria and relapsing fever Borrelia are infectious diseases characterized by fever, headache, myalgia, hepatosplenomegaly and tendency to relapse. Exflagellation of microgametocyte in malarial parasites is seen only in the definitive host, i.e., mosquitoes. Here we report an unusual case of a 23-year-old man who presented Plasmodium vivax infection with multiple exflagellated microgametes in the peripheral blood smear.

Background Chikungunya virus (CHIKV) is an emerging arbovirus, belonging to the Togaviridae family, Alphavirus genus, transmitted by Aedes spp. mosquitoes. Since 2007, two different CHIKV strains (E1-226A and E1-226V) have been responsible for outbreaks in European countries, including Italy, sustained by Ae. albopictus mosquitoes. Findings In this study, we assessed the susceptibility to the CHIKV E1-226V, strain responsible for the Italian 2007 outbreak, of eight Ae. albopictus populations collected in Northern, Central, Southern, and Island Italy, by experimental infections. Vector competence was evaluated by estimating infection, dissemination, and transmission rates (IR, DR, TR), through detection of the virus in the bodies, legs plus wings, and saliva, respectively. Additionally, vertical transmission was evaluated by the detection of the virus in the offspring. The results of our study demonstrated that the Italian populations of Ae. albopictus tested were susceptible to CHIKV infection, and can disseminate the virus outside the midgut barrier with high values of IR and DR. Viral infectious RNA was detected in the saliva of three populations from Central, Southern, and Island Italy, also tested for TR and population transmission rate (PTR) values. No progeny of the first and second gonotrophic cycle were positive for CHIKV. Conclusions This study strongly confirms the role of Ae. albopictus as a potential CHIKV vector in Italy. This may represent a threat, especially considering both the high density of this species, which is widespread throughout the country, and the increasing number of cases of imported arboviruses.

In August 2018 a Moroccan man living in Tuscany developed Plasmodium falciparum malaria. The patient declared having not recently visited any endemic country, leading to diagnostic delay and severe malaria. As susceptibility to P. falciparum of Anopheles species in Tuscany is very low, and other risk factors for acquiring malaria could not be completely excluded, the case remains cryptic, similar to other P. falciparum malaria cases previously reported in African individuals living in Apulia in 2017.

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.

Mosquitoes (Diptera: Culicidae) are the most important group of blood-sucking insects that are vectors of human diseases. This chapter focuses mainly on six species belonging to the Aedes, Culex and Anopheles genera, which, closely adapted to human habitats for thousands of years, have exploited human activities to spread and establish in areas far from their origin, becoming invasive. The mechanisms leading to the introduction and establishment of invasive mosquito species and the risk that they represent for human health in newly colonized areas are extensively described. In particular, this chapter focuses on the three powerful and widespread arbovirus disease vectors, Ae. aegypti, Ae. albopictus and Ae. japonicus, with shorter references to Ae. koreicus and other alien species recently recorded in Europe. The disease vectors belonging to the genus Culex are represented by the Cx. pipiens complex, whose history of travel between continents dates back centuries. The detailed story of the journey undertaken by species belonging to the Anopheles gambiae complex in the 19th century between Africa and Brazil represents the only significant example of invasion by Anophelinae mosquitoes. The last section of the chapter deals with the surveillance and control activities to be implemented in order to prevent the introduction and spread of invasive mosquito species.

Chromosome inversions have fascinated the scientific community, mainly because of their role in the rapid adaption of different taxa to changing environments. However, the ecological traits linked to chromosome inversions have been poorly studied. Here, we investigated the roles played by 23 chromosome inversions in the adaptation of the four major African malaria mosquitoes to local environments in Africa. We studied their distribution patterns by using spatially explicit modeling and characterized the ecogeographical determinants of each inversion range. We then performed hierarchical clustering and constrained ordination analyses to assess the spatial and ecological similarities among inversions. Our results show that most inversions are environmentally structured, suggesting that they are actively involved in processes of local adaptation. Some inversions exhibited similar geographical patterns and ecological requirements among the four mosquito species, providing evidence for parallel evolution. Conversely, common inversion polymorphisms between sibling species displayed divergent ecological patterns, suggesting that they might have a different adaptive role in each species. These results are in agreement with the finding that chromosomal inversions play a role in Anopheles ecotypic adaptation. This study establishes a strong ecological basis for future genome-based analyses to elucidate the genetic mechanisms of local adaptation in these four mosquitoes.