Assessment of the risk of malaria re-introduction in the Maremma plain (Central Italy) using a multi-factorial approach

In recent years, the increase in globalization [ 1 ], the rise in the average temperature of the earth together with an increasing frequency and intensity of extreme weather events, as storms, floods and droughts [ 2 , 3 ], and the environmental changes induced by human activities [ 4 ], have raised the concern about the possible introduction or reintroduction of Vector Borne Diseases in Countries where these were absent or eradicated [ 5 ]. These considerations, coupled with the recent spread of some mosquito vector borne diseases in Europe [ 6 , 7 ] and the increasing number of imported malaria cases recorded in the Continent [ 8 ] have renewed interest in the possible reintroduction of malaria in Southern Europe [ 7 – 9 ], particularly in the countries facing the Western Mediterranean Basin, where potential Anopheline vectors are still present [ 10 – 13 ]. Moreover, in recent years autochthonous malaria cases have been sporadically reported in Italy, France, Spain and Greece [ 14 – 20 ]. In 2005, a five-year study was implemented in Italy, as well as in other South European countries, with the aim to assess the status of the local potential malaria vectors and the possible re-introduction of malaria transmission [ 21 – 25 ]. In Italy, the selected study area was the Maremma plain, a region that was hyperendemic for malaria until 60 years ago [ 26 – 28 ] and that more recently was recognized as the major "at risk" area for the malaria reintroduction into Italy [ 14 , 29 , 30 ]. In Maremma, after the malaria eradication campaign (1947-1951), Anopheles labranchiae , the main endophilic vector of the Anopheles maculipennis complex was dramatically reduced in abundance. However, in subsequent years, the species has progressively re-colonized most of the area coming back to substantial densities [ 31 – 33 ]. This was mainly due to the introduction of intensive rice cultivation in the early 1970s. Since then, Maremma has been subjected to continuous entomological surveillance that was intensified after 1997, when an autochthonous Plasmodium vivax malaria case, transmitted by An. labranchiae , occurred in the Province of Grosseto [ 14 ]. The studies carried out in the area since eradication, provides a database that allowed a follow-up the history of malaria and its vectors in Maremma over the past 60 years. Starting from the findings of the most recent entomological and environmental studies [ 23 , 34 ], the present study was chosen to evaluate the malariogenic potential of the area using a multifactorial approach. The risk of malaria reintroduction was evaluated as the "malariogenic potential" of the study area by assessment of the three parameters that define it: a. receptivity of the area, given by the presence, distribution, seasonal abundance and bionomics of the potential vector; receptivity of the area, given by the presence, distribution, seasonal abundance and bionomics of the potential vector; b. susceptibility of the vector, that is its ability to become infected with Plasmodium vivax and Plasmodium falciparum ; susceptibility of the vector, that is its ability to become infected with Plasmodium vivax and Plasmodium falciparum ; c. vulnerability of the territory, that is the possible introduction of malaria reservoirs, given by the number of gametocyte carriers able to infect the vector and present in the study area during months favourable to malaria transmission. vulnerability of the territory, that is the possible introduction of malaria reservoirs, given by the number of gametocyte carriers able to infect the vector and present in the study area during months favourable to malaria transmission. Approaches to evaluate these parameters were: 1) Field collection of further entomological data (bionomics, distribution, abundance) for mosquitoes of the An. maculipennis complex; 2) investigation of seasonal dynamics of the vector through the implementation of a weather-based statistical dynamic model; 3) production of a distribution/predictive map of An. labranchiae across the study area; 4) evaluation of the length of the possible transmission season for P. vivax and P. falciparum ; 5) assessment of the vector competence of the species to P. falciparum by artificial infection; 6) evaluation of the vectorial capacity of An. labranchiae in the site where the species is most abundant; 7) risk assessment related to the possibility that the vector may feed on gametocyte carriers occasionally circulating in the study area. Maremma is a coastal plain of Central Italy, that covers about 5,500 km , extending along the Tyrrhenian coast for about 180 km from mid Tuscany (northern limit 43,3°N) to upper Latium (southern limit 42,17°N). Inland, Maremma extends from the sea to the pre-Apennine foothills, with a width ranging from 16 to 50 km. The territory includes different biotopes where natural and anthropic environments coexist. A wide sandy seashore and dunes, covered by typical Mediterranean bush, gives way to extensive pine woods. Large pastures alternate with intensively cultivated areas up to a range of hills that represents the inner Eastern limit of the study area. About 44,000 ha, nearly 10% of the whole surface of the Maremma plain, is included in protected reservation parks. The study area corresponds to the "core" of this region, shared between the administrative Provinces of Grosseto, and Siena (Tuscany) and Viterbo (Latium) (Figure 1 ). Ten collection sites were selected on the basis of their environmental characteristics, including three large rice cultivation areas, five rural areas with intensive farming and two protected coastal areas (Table 1 ). Map of the study area, the Maremma plain, with localization of the 10 sites considered in the study between 2005 and 2009 . Coastal marshes and the main rivers/streams are in dark grey. Triangles indicate the 4 sites selected for longitudinal surveillance (sites 1,3,4,6 in Table 1) visited fortnightly 6 months/year (May-October). Dots indicate the remaining 6 sites visited sporadically (1-4 times/season, in July-August). Collections of An. maculipennis s.l. mosquitoes were performed between 2005 and 2009 during 40 surveys. In particular, sites 1, 3, 4 and 6 were visited by 28 fortnightly surveys carried out from April 2005 to October 2006 [ 23 ], while in 2007-2009 these sites and the remaining ones were subject to sporadic surveys (1-4 times) in June-August. Mosquito larvae were collected by an enamel standard 500 ml dipper. The number of dipping stations and dips by station was adequate to the type and size of the breeding sites visited according to a standardized protocol [ 23 , 29 ]. Adult collections, using manual or battery-powered aspirators, were mainly targeted at resting females in animal shelters; other kinds of premises, such as haylofts, woodshed, fodder and tools depots represented less than 5% of the premises inspected. A minor fraction of An. maculipennis s.l. females were collected by CDC light/CO2 traps. Three night catches on human bait were seasonally carried out in late June, mid July and late August, between 2005 and 2007, respectively in Principina, S. Donato, and Val di Merse. Catches were performed as described in Romi et al. [ 29 ]. Mosquitoes were analyzed for species identification, blood meal source and age population structure as described in Di Luca et al. [ 23 ]. Previous entomological data used for comparisons are from the Operative Unit of Environmental Zoology, AUSL 9, Grosseto. A binomial model was developed for the less productive sites (4 and 6) categorizing the entomological data with the threshold of one adult ("A" absence category/"B" presence category), while the multinomial model, developed for the most productive site 1, was categorize with the thresholds of one and 60 adults ("A" absence category/"B" medium-low presence category/"C" medium-high presence category). The multi-logistic model input were derived from a principal component analysis that leads to an optimal dimensionality reduction of matrix predictors. Akaike Information Criterium (AIC) [ 35 ] was finally used for the statistical model parameters selection in order to optimize model performances and to give a better discrimination in the microclimatic variability among different sites. Concerning model selection the statistical function (i.e. STEPAIC) used is taken from MASS R package [ 36 , 37 ]. Weekly model outputs consist essentially on the occurrence probability associated to each adult abundance category, hence the forecasted category was chosen as the one with highest probability. The length of development season was estimated as the number of weeks between the first and the last week with a presence category (B or C category) along the year [ 38 – 41 ]. A verification procedure to assess the reliability of the models was implemented for each site using skill score indexes derived from contingency Tables with observed and forecasted values: the BIAS index, the POD index (Probability Of Detection) and the FAR index (False Alarm Ratio) [ 42 ]. The quantitative mapping of larvae/adult mosquito presence and abundance was implemented following the approach suggested by Tran et al. [ 43 ]. The logistic regression model for Anopheles hyrcanus in the Camargue region, that explained the presence of larvae as a function of biotope and distance to the nearest rice field, was modified in order to obtain a larval index (probability of observing one larva in a point of a biotope at least once during the mosquito season) consistent with the collection data reported in Table 1 . The logistic regression model for An. labranchiae , used in this study and implemented with Builder tool from ESRI ArcGIS™, consider also the distance from farm with livestock to the main biotopes as a new explanatory variable in addition to those used by Tran et al. Assuming that the abundance of adult mosquitoes is influenced by the presence of breeding sites in the surroundings, the adult index map was derived from the larval index map. Corine Land Cover 2000 (CLC 2000, produced by the European Environment Agency, EEA) spatial data sets for Italy (scale 1:100,000) was used to describe the environmental characteristics likely to influence the spatial distribution of An. labranchiae . Also a natural colour aerial photos (May 2007) with a spatial resolution of 1 metre was used to detect the main biotopes where An. labranchiae larvae and adults were collected, such as rice fields, reed beds, marshes, temporarily flooded rush wetland and clear water. A photo-interpretation was carried out through a workstation with ESRI ArcGIS™ software. Geographical database in shape-file format was used for localizing livestock and intensive cattle breeding farms in Grosseto Province [ 44 ]. The highest abundance of An. labranchiae was assumed to be related to rice paddies, and thus the distance to the nearest rice field was computed for each pixel using Geographic Information System (GIS) functionality. Since Grosseto Airport weather station (42.75°N-11.07°E, 7 m. a.s.l.) is the climate reference centre for the World Meteorological Organization [WMO], meteorological data from that station were considered representative of the whole study area. Thermo-pluviometric diagram with monthly temperature and respectively over a and over the study were with the temperature for development of P. vivax and P. falciparum , and respectively [ ]. The average and on a and seasonal basis of temperature and were for and in order to the possible from a point of (Table 2 ). evaluation of the Plasmodium possible transmission season along the years 2005 and for the between 2005 and 2009 and for was also performed through the index [ 25 , 37 ]. on the monthly of of the index to into possible transmission along the this index parameters as the temperature for the development of the the vector, as reported monthly and by the where is with a temperature of and respectively for P. vivax and P. falciparum , R is the and is potential with the of [ ] as a function of temperature and The index that a transmission risk when its is or than that is the for one Plasmodium Field of An. labranchiae collected in site 1 (Figure 1 , Table 1 were to the to artificial blood with of the P. falciparum in and of and artificial blood were performed following described in et al. [ ]. A of Anopheles was used as a Mosquitoes were on 8 and to and in the each An. labranchiae females and the An. were to blood The infected blood falciparum with and human were in a at An. labranchiae were a blood meal on the when females were at 26 and were with 10% until 8 or In the the of was carried out at the et in using the from the was performed by a according to et al. [ ]. of infected females were used for species by [ ]. capacity was according to the [ 50 ] by [ ]. The for were evaluated as the human by night catches on human the human blood index by the of the blood meal of the females collected early in the in different premises and the by [ ]. The such as the length of the of P. falciparum and P. vivax and the of were according to [ 50 ]. The An. labranchiae was estimated by different in addition to the the and the index vector selection of a than other was by the of females on a given by the that represented in the of and at the site [ ]. indicate a and indicate in of other is as the observed of females on a animal with to one by the of on these two [ ]. This index was by in that such as their size and their and spatial with the mosquito 1 on the two while or indicate a or increase in on the first to the were for each of The possible between abundance and size of the fraction during night catches was also evaluated by the statistical the data recorded in the area of site 1 with those from site 3 over a of 14 years A of of An. labranchiae (i.e. the of the number of those was also by the analysis of the data from human bait performed and in The number of gametocyte carriers that may have been circulating in the territory during the favourable to malaria transmission was by the analysis of the cases of imported malaria in Italy in by the at di those reported from into the study area. are and by for the malariogenic potential of the study area. of a of females to the An. maculipennis complex considered in this were and at species at different of and abundance (Table 1 An. labranchiae occurred in the study sites where represents the species of the maculipennis with the of site 3 and the rice of Val di and the farm of in an area over where its for and An. maculipennis of the different climate [ 23 , 34 ]. The rice of the coastal plain (sites the most productive areas of An. labranchiae where represents of the species to the A of labranchiae with of abundance was recorded in the remaining study where changes in occurred during the last three have to the territory less favourable to the development of A of recent findings with those for the a reduction of the abundance of An. . resting females in the study area of about The matrix from the of the weather-based statistical model that variability in the last 7 and are the most in abundance of An. labranchiae adult females for the three collection the other meteorological in less to model for each abundance category, for the multinomial or the binomial model, a capacity to absence from presence in the three the POD index was and FAR performances still were for in site 1 the medium-low abundance from medium-high abundance POD to and FAR to The BIAS index was in cases to 1, hence be that or of the model is the between and observed of development season length of An. labranchiae adult females (Table 3 in a for site 6 in 2005 and while for site 4 the length three weeks than the observed one in 2005 and for site 1 two weeks in 2005 and three weeks in The two distribution of adult An. labranchiae over the study area are in 2 . The An. labranchiae larval index on of Anopheles . that the occurrence of the species on rice areas with a probability of presence The adult index, for each site as the of the larval index a size 500 to the collection was to the observed abundance of An. labranchiae , in order to the The between the adult index and the observed number of An. labranchiae was for a of 2000 m. and used to map the abundance of An. labranchiae adult over the study area (Figure ). In order to these the adult indexes by the map in the sites reported in Table 1 were with the of females abundance collected in in the sites (Figure ). Field collected data well with those by the with the of site 10 where the large surface of the to the adult and of site 3 di where the between the of abundance and is the map considered the of An. maculipennis . while the to the of An. labranchiae among the other species of the complex in that area In , the map of the An. labranchiae adult population in Grosseto also the livestock presence, an presence of areas with densities than particularly in the weather-based distribution of adult Anopheles labranchiae in Maremma, Central Italy . Adult distribution is related to a larval index by of longitudinal surveillance data from the 4 sites represented by (sites 1,3,4,6 in Map A where and the between spatial of abundance and its to the most breeding sites of the whole study area. the adult abundance recorded in the remaining 6 sites visited sporadically (Table 1) and used to the map. Map also into livestock densities as a for each pixel of the adult distribution map of the Grosseto Province by the The of the monthly temperature that the favourable transmission for the Plasmodium species for one during to In during the of the favourable transmission from to April for P. vivax and to for P. falciparum (Figure 3 ). of the diagram of the Grosseto for and . and the monthly of and the monthly temperature for and respectively the temperature for the development of vivax and P. falciparum that are respectively and Plasmodium potential transmission risk evaluated through the index [ 25 , 37 ] to be the a potential risk during for P. vivax in 2005 or 2006 as well as for the while for the the index remaining the for P. falciparum the index the threshold in 2006 ). index monthly diagram for malaria transmission in Maremma region . the of transmission risk for Plasmodium vivax A and for P. falciparum in 2005, 2006 and the one in and Concerning the rice field biotope be that is considered and to one in of the length of the possible transmission season) since artificial in rice is continuous during and index corresponds to with a temperature of for P. vivax and for P. falciparum . the basis of this the favourable transmission risk for P. vivax (Figure between and 2005 and for or October for to the Concerning P. falciparum , favourable transmission risk (Figure between July and August, also or to the threshold in index monthly diagram the of malaria transmission in rice in 2005, 2006 . the of transmission risk for Plasmodium vivax A and for P. falciparum in 2005, 2006 and the one in and index is to when the is to The susceptibility of An. maculipennis . population from site 1 to P. falciparum was by of of artificial with a of In resting females were collected on the field and three were In 25 out of mosquitoes until 8 an of and a number of in the (Table 4 ). The of species of the infected mosquitoes were: An. labranchiae and one Anopheles . In were performed using resting out of the mosquitoes in were infected in one was observed a The was and the number of was (Table 4 ). was possible to the of with and in the for in the including the with were An. labranchiae , An. and 21 of An. labranchiae , one with and one in the were to be In and some of first of An. labranchiae were also carried out infected mosquitoes were in these the than 5% of the field collected females an infected blood and of these mosquitoes In the An. infected with ranging from to A study on the assessment of a vectorial capacity of An. labranchiae in Maremma was performed with the vector population of site 1, the of Principina, where the highest abundance of the species was the species of the This was the site for was possible to the human blood index A of the entomological for is given in Table 5 . An. labranchiae was the mosquito recorded in 6 of collection from to August, The of An. labranchiae from 6 to The was by the blood analysis of females collected in resting of the human blood and blood [ 23 ]. The of An. labranchiae from in site 1 as and 2005 and 10% and 2006 in site 1 [ 23 ]. this of the probability of of An. labranchiae was In the 2005 the between and with an of for P. falciparum ranging between and and from and for P. vivax . In the between and with of for P. falciparum between and and between and for P. vivax . this in 2005 the for P. falciparum from to and from to for P. vivax ; in 2006 from to for P. falciparum and from to for P. vivax . The highest were in July 2005 for the Plasmodium the of the and its of was by the of two more and the animal in the study the for this the among a for the other species (Table 6 for cattle and A of an of A further evaluation was performed using a modified into the size of each and the or occurrence of An. labranchiae mosquitoes and during the The of human with the of each a of An. labranchiae for with to the cattle and the the for and that these were the source of blood with to the The average of the An. labranchiae for the three years was and a of the potential vector for are (i.e. further of the more of An. labranchiae with to other species of the An. maculipennis the between data for of resting females abundance from and and number of in sites 1 and 6 was at in site 1 (Figure where An. labranchiae is the while in site 3 (Figure where species is An. maculipennis the was diagram between adult resting females abundance and number of in two sites of Maremma over a of 14 years . Dots the number of females collected animal in the of the site of where species is Anopheles and of the Val di site where species is An. maculipennis in is at at A possible introduction of gametocyte carriers from malaria areas able to infect the potential during the season favourable to malaria was evaluated (Table 7 ). In the 10 cases from gametocyte carriers coming from mainly from were due to P. falciparum P. vivax and Plasmodium cases were considered due to P. falciparum , one to P. vivax and two to P. An. labranchiae occurred in the season favourable to malaria transmission