**2. Malaria**

484 Current Topics in Tropical Medicine

*Ae. vexans* All over Europe Indigenous WN, TAH, Tularaemia (TU),

*An. claviger s.l.* All over Europe Indigenous MAL, WN, Batai (BAT), TAH,

*An. maculipennis* s.l. All over Europe Indigenous MAL, WN, BAT, TAH, MYX,

*Cx. pipiens* s.l. All over Europe Indigenous WN, SIN, Usutu (USU), TAH,

Exotic (first reported in Italy in 1996)

*Oc. caspius* All over Europe Indigenous WN, TAH, MYX, TU, DF

Exotic (recently imported)

Exotic (not yet known as established)

Table 1. Mosquito vectors in Europe with indication of distribution, indigenous or exotic

*An. plumbeus* All over Europe Indigenous MAL, WN, DF

*An. sergentii* Sicily (Italy) Indigenous MAL

DF

Indigenous DEN, YF, CHIK, AMAL

MYX, Anaplasmosis (ANA),

Borreliosis (BO),

TU, DF

TU, DF

AMAL, DF

WEE, EEE

WN, JE, SLE, LACE, MVE,

WN, JEV, SLE, LACE, EEE

DEN, YF, WN, SLE, LACE,

WEE, EEE, VEE, JC

Indigenous Malaria (MAL)

Indigenous MAL, DF

Vector species Distribution Indigenous/exotic Vectorial capacity

*Ae. vittatus* Spain, Portugal,

*An. algeriensis* Mediterranean

*An. superpictus* Southeastern

*Oc. atropalpus* Italy,

*Oc. japonicus* France,

*Oc. triseriatus* Intercepted in a

Europe

France, The Netherlands.

Belgium, Switzerland, Germany

batch of used tyres imported from Louisiana

(USA) to France in 2004

status and vectorial capacity in each case.

France, Italy

Eastern Europe, Central Europe, United Kingdom

area,

Malaria was a widespread disease in the whole of Europe until the second half of 20th century. The anthroponosis, often called "marsh fever" in the past, was particularly devastating between XVI and XIX centuries in Southern Europe due to the boom of irrigation techniques based on long flooding periods (e.g. rice fields). Several environmental modifications (mainly the drainage of swamps, moats, ditches and other stagnant waters), but particularly the availability of efficient synthetic antimalarial drugs and improved mosquito control activities including DDT spraying after World War II, have led to the disappearance of malaria from Europe (Bruce-Chwatt & de Zulueta, 1980). However, although *Anopheles* populations were significantly reduced by different control methods, in most cases, the vectors were not eradicated.

Today malaria annually affects 500 million people and threatens directly or indirectly 40% of world population (World Health Organization [WHO], 2007). However it is well known that these morbidity and mortality data show an asymmetric distribution, mainly depending on the economical, social and sanitary level of each country or region. The disease is endemic in much of Africa and several countries of Asia, Central America and South America. In Europe, the cycles of malaria transmission are relatively common in Georgia, Azerbaijan, Kyrgyzstan, Tajikistan, Uzbekistan and Turkey (WHO, 2010). This mosquito-borne parasitaemic disease is caused by protozoa of the genus *Plasmodium*. Although the simian parasite *Plasmodium knowlesi* (Knowles and Das. Gupta 1932) has been found recently as a cause of human malaria in Southeastern Asia (Luchavez et al., 2008), other four plasmodia species are the most recognized to infect humans in nature conditions: *Plasmodium falciparum* (Welch, 1897), *Plasmodium vivax* (Grassi & Feletti, 1890), *Plasmodium malariae* (Feletti & Grassi, 1889) and *Plasmodium ovale* (Stephens, 1922). About 90% of malaria mortality is caused by tropical strains of *P. falciparum* (most pathogenic species), which is also the species of *Plasmodium* most frequently imported to Europe (European Network on Imported Infectious Disease Surveillance [TropNetEurop], 2010). Furthermore, *P. vivax* shows the largest distribution range because it may also develop in temperate climates, being consequently the only species currently present in the cycles of transmission in Europe. Finally, *P. malariae* and *P. ovale* are characterized by its narrow distribution range and low parasitemia. Regarding to malaria vectors, there are about 40 *Anopheles* species with an important role in disease transmission (Kiszewski, 2004).

#### **2.1 Malariogenic potential of Europe**

The increasing of imported malaria cases in last decades, together with the high presence of anophelines in many Southern Europe regions (Romi et al*.,* 1997; Ponçon et al., 2007; Bueno Marí & Jiménez Peydró, 2010a), has enabled the appearance of several autochthonous malaria cases, as recently has occurred in countries like Italy (Baldari et al., 1998), Greece (Kampen et al., 2002), France (Doudier et al., 2007) or Spain (Santa-Olalla Peralta et al., 2010). This situation forces us to investigate the possible reemergence of malaria in the current context of global change. One of the best methods to deep into the knowledge of possible malaria reemergence is the study of the malariogenic potential, which can be analyzed from the study of the receptivity, infectivity and vulnerability parameters (Romi et al., 2001; Bueno Marí & Jiménez Peydró, 2008).

Re-Emergence of Malaria and Dengue in Europe 487

Argelia (non demonstrated vector in Europe)

Northern Europe, Central Europe, Eastern Europe, Mediterranean Europe

Eastern Mediterranean countries, Central Asia

Asia (as An. hyrcanus s.l.)

(Peninsular Italy, Sardinia and Sicily), Southeastern Spain (disappeared since 1973)

Coastal areas in the Balkans, Asia Minor, Northern Iran


Eastern Europe

Anopheles Species European distribution Malaria outbreaks

Ger, Aust, Ital, Sard, Sic, Croa, Alb, Gree, Turk, Hung, Bulg, Moldv, Ukr, EurRus, Est

Belg, Neth, Ger, Aust, Czech, Slovk, Pol, Switza, Ital, Ser-Mon, Croa, Bosn, Slovn, Mace, Hung, Rom, Bulg, Moldv, Ukr, Bela, EurRus, Lith, Latv

*An. beklemishevi* Swe, Fin, EurRus - *An. cinereus* Spain, Port -

> Spain, Port, Belg, Neth, Lux, Ger, Aust, Czech, Slovk, Pol, Switz, Ital, Sic, Ser-Mon, Croa, Bosn, Slovn, Mace, Alb, Gree, Turk, Cypr, Hung, Rom, Bulg, Moldv, Ukr, Bela, EurRus, Lith, Latv, Est

*An. daciae* <sup>b</sup> Brit, Rom -

Mon, Croa, Mace, Alb, Gree, Turk, Hung, Rom, Bulg, Moldv, Ukr, EurRus

Belg, Neth, Lux\*, Ger, Aust, Czech, Slovk, Pol, Switza, Ital, Sic, Ser-Mon, Croa, Bosn, Slovn, Mace, Alb, Gree, Turk, Hung, Rom, Bulg, Moldv, Ukr, Bela, EurRus, Lith, Latv, Est

Gree, Turk, Bulg

Belg, Neth, Ger, Aust, Czech, Slovk, Pol, Switza, Ital, Ser-Mon, Croa, Bosn, Slovn, Mace, Alb, Gree, Hung, Rom, Bulg, Moldv, Ukr, Bela, EurRus, Lith, Latv, Est

*An. melanoon c* Fra, Cors, Spain, Ital, Rom, EurRus -

*An. labranchiae* Cors, Ital, Sard, Sic, Croa France (Corsica), Italy

*An. algeriensis* Brit, Ire, Fra, Cors, Spain, Bala, Port,

*An. atroparvus* Brit, Ire\*, Swe, Den, Fra, Spain, Port,

*An. claviger* Brit, Ire, Nor, Swe, Den, Fra, Cors,

*An. hyrcanus* Fra, Cors, Spain, Ital, Sard, Sic, Ser-

*An. maculipennis* Nor, Swe, Den, Fra, Cors, Spain, Port,

*An. marteri* Cors, Spain, Port, Ital, Sard, Sic, Alb,

*An. messeae* Brit, Irea, Nor, Swe, Den, Fra, Cors,

### **2.1.1 Receptivity**

Receptivity could be analyzed by the presence, density, and biological characteristics of vectors. At respect, the estimation of the Vectorial Capacity (VC) is postulated as a very useful tool to assess the receptivity of a determined territory in a concrete moment (Carnevale & Robert, 2009). The VC could be estimated by the MacDonald formula (MacDonald, 1957) according to the modifications proposed by Garrett-Jones (1964):

$$\mathsf{VC} = ma 2 \; p^n \; / \text{ -} \ln \; p^n$$

Where, m represents the relative vector density (number of vectors per man), *a* refers to human-biting frequency (number of human blood meals per vector and per day), *p* is the daily survival rate (life expectancy of the female mosquito) and *n* alludes to duration of the sporogonic cycle (length in days of the latent period of the parasite in the mosquito, i.e. extrinsic incubation cycle). It is important to note that *ma* is usually measured by collecting mosquitoes during an entire night using human bait. Consequently VC could be defined as the future daily sporozoite inoculation rate arising from a currently infective human case, on the assumption that all female mosquitoes biting that person become infected (Githeko, 2006). Of course VC changes from site to site, from vector to vector, and within and between transmission seasons.

#### **2.1.1.1 Malaria receptivity in Southern Europe**

Because of climatic conditions, the Southern Europe represents the territory of the Old Continent where disease cycles can be completed more likely. In terms of receptivity, of twenty species of *Anopheles* described in Europe twelve are confined in its distribution to Southern areas (Table 2). In the Iberian Peninsula rice cultivation was clearly associated with malaria endemicity until the beginning of the 20th century (Cambournac & Hill, 1938; Cambournac, 1939, Blázquez, 1974; Bueno Marí & Jiménez Peydró, 2010b). In these larval biotopes the species *Anopheles atroparvus* and, to a much lesser extent and only in the more arid areas, *Anopheles labranchiae* were supposed to be the major malaria vectors (Bruce-Chwatt & de Zulueta, 1977), although some other species, such as *Anopheles maculipennis* or *Anopheles claviger* may locally also have contributed to disease transmission (Bueno Marí, 2010). Currently *An. atroparvus* remains widespread in rice fields and other potential *Anopheles* breeding sites of Portugal and Spain (Capinha et al. 2009; Sainz-Elipe et al. 2010), since the most important western Mediterranean malaria vector *An. labranchiae* is considered dissapeared. *An. labranchiae* was found to be abundant in a restricted area of the contiguous Alicante and Murcia Provinces (South-eastern Spain) in 1946 (Clavero & Romeo Viamonte, 1948), but had disappeared by 1973 (Blázquez & de Zulueta, 1980) probably due to abandonment of rice cultivation in this area (Eritja et al., 2000). Recent surveys carried out in this area have revealed again the absence of *An. labranchiae* as well as high populations of the secondary vector *Anopheles algeriensis* also characterized by high domiciliation degrees (Bueno Marí, 2011). This was the only area where *An. labranchiae* has been able to establish itself in the Iberian Peninsula (Blázquez & de Zulueta, 1980). Though abundant along the African coastline between Ceuta and Tangiers, *An. labrachiae* has been unable to obtain a toe-hold in 15 km distant coastal plains of southern Spain, where rice fields support large populations of *An. atroparvus* (Ramsdale & Snow, 2000). It is important to note that the most important vector of the Iberian Peninsula *An. atroparvus* is suspected of being the vector of an autochthonous case of *Plamodium vivax* which recently occurred in Northeastern Spain (Santa-Olalla Peralta et al., 2010) and even also in other case of *Plamodium ovale* which happened in Central Spain, although airport malaria cannot be discarded in this last case due to the proximity of the patient's residence to two international airports (Cuadros et al., 2002).

Receptivity could be analyzed by the presence, density, and biological characteristics of vectors. At respect, the estimation of the Vectorial Capacity (VC) is postulated as a very useful tool to assess the receptivity of a determined territory in a concrete moment (Carnevale & Robert, 2009). The VC could be estimated by the MacDonald formula

VC = *ma*2 *pn* / -ln *p* Where, m represents the relative vector density (number of vectors per man), *a* refers to human-biting frequency (number of human blood meals per vector and per day), *p* is the daily survival rate (life expectancy of the female mosquito) and *n* alludes to duration of the sporogonic cycle (length in days of the latent period of the parasite in the mosquito, i.e. extrinsic incubation cycle). It is important to note that *ma* is usually measured by collecting mosquitoes during an entire night using human bait. Consequently VC could be defined as the future daily sporozoite inoculation rate arising from a currently infective human case, on the assumption that all female mosquitoes biting that person become infected (Githeko, 2006). Of course VC changes from site to site, from vector to vector, and within and between

Because of climatic conditions, the Southern Europe represents the territory of the Old Continent where disease cycles can be completed more likely. In terms of receptivity, of twenty species of *Anopheles* described in Europe twelve are confined in its distribution to Southern areas (Table 2). In the Iberian Peninsula rice cultivation was clearly associated with malaria endemicity until the beginning of the 20th century (Cambournac & Hill, 1938; Cambournac, 1939, Blázquez, 1974; Bueno Marí & Jiménez Peydró, 2010b). In these larval biotopes the species *Anopheles atroparvus* and, to a much lesser extent and only in the more arid areas, *Anopheles labranchiae* were supposed to be the major malaria vectors (Bruce-Chwatt & de Zulueta, 1977), although some other species, such as *Anopheles maculipennis* or *Anopheles claviger* may locally also have contributed to disease transmission (Bueno Marí, 2010). Currently *An. atroparvus* remains widespread in rice fields and other potential *Anopheles* breeding sites of Portugal and Spain (Capinha et al. 2009; Sainz-Elipe et al. 2010), since the most important western Mediterranean malaria vector *An. labranchiae* is considered dissapeared. *An. labranchiae* was found to be abundant in a restricted area of the contiguous Alicante and Murcia Provinces (South-eastern Spain) in 1946 (Clavero & Romeo Viamonte, 1948), but had disappeared by 1973 (Blázquez & de Zulueta, 1980) probably due to abandonment of rice cultivation in this area (Eritja et al., 2000). Recent surveys carried out in this area have revealed again the absence of *An. labranchiae* as well as high populations of the secondary vector *Anopheles algeriensis* also characterized by high domiciliation degrees (Bueno Marí, 2011). This was the only area where *An. labranchiae* has been able to establish itself in the Iberian Peninsula (Blázquez & de Zulueta, 1980). Though abundant along the African coastline between Ceuta and Tangiers, *An. labrachiae* has been unable to obtain a toe-hold in 15 km distant coastal plains of southern Spain, where rice fields support large populations of *An. atroparvus* (Ramsdale & Snow, 2000). It is important to note that the most important vector of the Iberian Peninsula *An. atroparvus* is suspected of being the vector of an autochthonous case of *Plamodium vivax* which recently occurred in Northeastern Spain (Santa-Olalla Peralta et al., 2010) and even also in other case of *Plamodium ovale* which happened in Central Spain, although airport malaria cannot be discarded in this last case due to the proximity of the

(MacDonald, 1957) according to the modifications proposed by Garrett-Jones (1964):

**2.1.1 Receptivity** 

transmission seasons.

**2.1.1.1 Malaria receptivity in Southern Europe** 

patient's residence to two international airports (Cuadros et al., 2002).


Re-Emergence of Malaria and Dengue in Europe 489

Until the beginning of dichlorodiphenyltrichloroethane (DDT) application, the main malaria vectors in Italy were *An. superpictus* as well as two species of the *Anopheles maculipennis* complex: *An. labranchiae* and *An. sacharovi* (Hackett & Missiroli 1935). Despite *An. labranchiae* used to breed in various types of waters, such as marshes, streams, small pools or irrigation channels, the rice fields established in the 1970s currently represent its most important larval habitats in Central Italy (Bettini et al., 1978; Romi et al., 1992). Even in Western province of Grosseto *An. labranchiae* has replaced *Anopheles melanoon*, species that in 1970 represented for 100% of the anophelines fauna (Majori et al., 1970). Precisely in Grosseto region occurred the last autochthonous malaria case in Italy in August 1997 (Baldari et al., 1998). Nowadays of the anopheline species that have been vectors of malaria in Italy, only *An. labranchiae* and *An. superpictus* are still present in epidemiologically relevant densities (Romi et al., 1997). Moreover *An. atroparvus* is also present in Italy at low densities and *An. sacharovi* is currently considered disappeared, since last specimens of the vector were found 50 years ago

In Balkan countries (Bulgaria, Romania, Croatia, Serbia, Bosnia-Herzegovina, Montenegro and Albania, among others) the species *An. sacharovi* used to be the main malaria vector in coastal areas while *An. superpictus* and *An. maculipennis* were the primary vectors in inland areas due to the specific adaptations of their preimaginal stages (Hackett, 1937; Hadjinicolaou & Betzios, 1973; Bruce-Chwatt & de Zulueta, 1980). Larvae of *An. sacharovi* are tolerant against brackish water but not against salt water. On the other hand *An. superpictus* breeds in slowly flowing waters in hilly areas while *An. maculipennis* breeds in stagnant inland waters (Jetten & Takken, 1994). However, when sporadically *An. maculipennis* has colonized coastal areas of Balkans, Asia Minor and Northern Iran, it has also showed an important role in malaria transmission (Postiglione et al., 1973; Zaim, 1987; Manouchehri et al., 1992; Schaffner et al., 2001). Of the three most important vectors of Balkans, *An. superpictus* was never collected in Romania. Therefore in this country in addition to *An. sacharovi* and *An. maculipennis*, also *Anopheles messeae* and *An. atroparvus* have contributed to the endemism of malaria. Generally *An. messeae* has played a prominent role as a malaria vector in the Danube Valley and Delta, while *An. maculipennis* was mainly responsible for malaria transmission in the Romanian plains and *An. sacharovi* and *An. atroparvus* have been primary vectors at the Black Sea coast (Zotta, 1938; Zotta et al., 1940; Ciuca, 1966). All these issues represent the concept of "malaria stratification", which indicates a good relation between the distribution of the different anophelines species and the great "malaria geographic lines" (Nicolescu, 1996). Moreover a new species of the *An. maculipennis* complex, named *Anopheles daciae*, was recently first described in Romania (Nicolescu et al., 2004). It seems likely that *An. daciae* could be widespread in Eastern Europe and the Balkan States, and also could be responsible for malaria transmission in these regions that is

In order of relevance, *An. sacharovi*, *An. superpictus* and *An. maculipennis* were considered the main malaria vectors in Greece (Belios, 1955, 1978). During the recent years several autochthonous cases of *P. falciparum*, *P. malariae* and *P. vivax* have been diagnosed in Northern Greece (Kampen et al., 2002). At respect, it is important to note the proximity of this region to an unstable malaria country as Turkey. In Turkey malaria is still one of the most important vector-borne diseases in Turkey (Kasap et al., 2000; Alten et al., 2003), even remaining some endemic areas with hundreds of vivax cases yearly. The most important vectors in Turkey are *An. sacharovi* and *An. superpictus* (Kuhn et al., 2002), taking *An. maculipennis*, *An. claviger* and *Anopheles hyrcanus* a secondary role in malaria transmission.

(Sepulcri, 1963).

currently attributed to *An. messeae*.


*Note 1*: Countries with anophelines records considered as doubtful or sporadic were not included. If it is thought that the species has been eradicated, the country is also not listed. *Note 2*: Brit (Britain), Ire (Ireland), Nor (Norway), Swe (Sweden), Den (Denmark), Fra (France), Cors (Corsica), Spain, Bala (Balearic Islands), Port (Portugal), Belg (Belgium), Neth (Netherlands), Lux (Luxemburg), Ger (Germany), Aust (Austria), Czech (Czech Republic), Slovk (Slovakia), Pol (Poland), Switz (Switzerland), Ital (Italy), Sard (Sardinia), Sic (Sicily), Malt (Malta), Ser-Mon (Serbia-Montenegro), Croa (Croatia), Bosn (Bosnia), Slovn (Slovenia), Mace (Macedonia), Alb (Albania), Gree (Greece), Turk (Turkey), Cypr (Cyprus), Hung (Hungary), Rom (Romania), Bulg (Bulgaria), Moldv (Moldavia), Ukr (Ukraine), Bela (Belarus), EurRus (Eropean Russia), Lith (Lithuania), Latv (Latvia), Est (Estonia).

a Records referred to *Anopheles maculipennis* s.l. b Species recently described by molecular and morphological techniques.

c There is confusion with these two species.

d Present in Asiatic Turkey.

Table 2. *Anopheles* species with endemic presence in Europe and indication of historical data about its vectorial role (Ramsdale & Snow, 2000; Schaffner et al., 2001; Beck et al., 2003; Nicolescu et al., 2004; Linton et al., 2005; Becker et al., 2010; European Mosquito Taxonomists [MOTAX], 2010).

In France, the same two species mentioned above for the Iberian Peninsula, are also considered to be primary malaria vectors because of their abundance and their potential anthropophily: *An. atroparvus* in continental France and *An. labranchiae* in Corsica. In a former malaria-endemic area of Southern France, intensive samplings conducted recently in rice fields showed that *Anopheles hyrcanus* seems to be the only potential vector likely to play a role in malaria transmission in view of its abundance and anthropophily (Ponçon et al., 2007). Since 1994 several cases of vivax and falciparum malaria with no history of international travels, blood transfusion or injection drug use have been reported in Southern France (Delmont et al., 1994; Baixench et al., 1998; Doudier et al., 2007). In Corsica, where *An. labranchiae* still present in high densities in different regions (Toty et al., 2010), autochthonous *P. vivax* malaria transmission has been diagnosed, probably via the bite of a local *Anopheles* mosquito infected with *P. vivax* from a patient who had acquired infection in Madagascar (Armengaud et al., 2006). The second most important malaria vector of Corsica, *Anopheles sacharovi*, has not been detected in the island since 2002 (Toty et al., 2010).

England, Germany, Caucasus

Near East

Albania, Greece

Middle East

*An. multicolor* Spain - *An. petragnani* Fra, Cors, Spain, Port, Ital, Sard, Sic -

> Port, Belg, Neth, Lux, Ger, Aust, Czech, Slovk, Pol, Switz, Ital, Sic, Ser-Mon, Croa, Bosn, Slovn, Mace, Alb, Gree, Turk, Hung, Rom, Bulg, Ukr, Bela, EurRus, Lith, Est

*An. pulcherrimus d* Turk Middle East

Gree, Turk, Bulg, EurRus

Alb, Gree, Turk, Bulg, EurRus

Alb, Gree, Turk, Bulg, EurRus

*An. sergentii* Sic Mediterranean Africa

*Note 1*: Countries with anophelines records considered as doubtful or sporadic were not included. If it is thought that the species has been eradicated, the country is also not listed. *Note 2*: Brit (Britain), Ire (Ireland), Nor (Norway), Swe (Sweden), Den (Denmark), Fra (France), Cors (Corsica), Spain, Bala (Balearic Islands), Port (Portugal), Belg (Belgium), Neth (Netherlands), Lux (Luxemburg), Ger

(Germany), Aust (Austria), Czech (Czech Republic), Slovk (Slovakia), Pol (Poland), Switz (Switzerland), Ital (Italy), Sard (Sardinia), Sic (Sicily), Malt (Malta), Ser-Mon (Serbia-Montenegro), Croa (Croatia), Bosn (Bosnia), Slovn (Slovenia), Mace (Macedonia), Alb (Albania), Gree (Greece), Turk (Turkey), Cypr (Cyprus), Hung (Hungary), Rom (Romania), Bulg (Bulgaria), Moldv (Moldavia), Ukr (Ukraine), Bela

Table 2. *Anopheles* species with endemic presence in Europe and indication of historical data about its vectorial role (Ramsdale & Snow, 2000; Schaffner et al., 2001; Beck et al., 2003; Nicolescu et al., 2004; Linton et al., 2005; Becker et al., 2010; European Mosquito

In France, the same two species mentioned above for the Iberian Peninsula, are also considered to be primary malaria vectors because of their abundance and their potential anthropophily: *An. atroparvus* in continental France and *An. labranchiae* in Corsica. In a former malaria-endemic area of Southern France, intensive samplings conducted recently in rice fields showed that *Anopheles hyrcanus* seems to be the only potential vector likely to play a role in malaria transmission in view of its abundance and anthropophily (Ponçon et al., 2007). Since 1994 several cases of vivax and falciparum malaria with no history of international travels, blood transfusion or injection drug use have been reported in Southern France (Delmont et al., 1994; Baixench et al., 1998; Doudier et al., 2007). In Corsica, where *An. labranchiae* still present in high densities in different regions (Toty et al., 2010), autochthonous *P. vivax* malaria transmission has been diagnosed, probably via the bite of a local *Anopheles* mosquito infected with *P. vivax* from a patient who had acquired infection in Madagascar (Armengaud et al., 2006). The second most important malaria vector of Corsica,

*Anopheles sacharovi*, has not been detected in the island since 2002 (Toty et al., 2010).

*An. plumbeus* Brit, Ire, Swe, Den, Fra, Cors, Spain,

*An. sacharovi* Cors, Ser-Mon, Croa, Mace, Alb,

*An. subalpinus c* Fra, Cors, Port, Ser-Mon, Croa, Mace,

*An. superpictus* Cors, Ital, Sic, Ser-Mon, Croa, Mace,

(Belarus), EurRus (Eropean Russia), Lith (Lithuania), Latv (Latvia), Est (Estonia).

a Records referred to *Anopheles maculipennis* s.l. b Species recently described by molecular and morphological techniques.

c There is confusion with these two species.

d Present in Asiatic Turkey.

Taxonomists [MOTAX], 2010).

Until the beginning of dichlorodiphenyltrichloroethane (DDT) application, the main malaria vectors in Italy were *An. superpictus* as well as two species of the *Anopheles maculipennis* complex: *An. labranchiae* and *An. sacharovi* (Hackett & Missiroli 1935). Despite *An. labranchiae* used to breed in various types of waters, such as marshes, streams, small pools or irrigation channels, the rice fields established in the 1970s currently represent its most important larval habitats in Central Italy (Bettini et al., 1978; Romi et al., 1992). Even in Western province of Grosseto *An. labranchiae* has replaced *Anopheles melanoon*, species that in 1970 represented for 100% of the anophelines fauna (Majori et al., 1970). Precisely in Grosseto region occurred the last autochthonous malaria case in Italy in August 1997 (Baldari et al., 1998). Nowadays of the anopheline species that have been vectors of malaria in Italy, only *An. labranchiae* and *An. superpictus* are still present in epidemiologically relevant densities (Romi et al., 1997). Moreover *An. atroparvus* is also present in Italy at low densities and *An. sacharovi* is currently considered disappeared, since last specimens of the vector were found 50 years ago (Sepulcri, 1963).

In Balkan countries (Bulgaria, Romania, Croatia, Serbia, Bosnia-Herzegovina, Montenegro and Albania, among others) the species *An. sacharovi* used to be the main malaria vector in coastal areas while *An. superpictus* and *An. maculipennis* were the primary vectors in inland areas due to the specific adaptations of their preimaginal stages (Hackett, 1937; Hadjinicolaou & Betzios, 1973; Bruce-Chwatt & de Zulueta, 1980). Larvae of *An. sacharovi* are tolerant against brackish water but not against salt water. On the other hand *An. superpictus* breeds in slowly flowing waters in hilly areas while *An. maculipennis* breeds in stagnant inland waters (Jetten & Takken, 1994). However, when sporadically *An. maculipennis* has colonized coastal areas of Balkans, Asia Minor and Northern Iran, it has also showed an important role in malaria transmission (Postiglione et al., 1973; Zaim, 1987; Manouchehri et al., 1992; Schaffner et al., 2001). Of the three most important vectors of Balkans, *An. superpictus* was never collected in Romania. Therefore in this country in addition to *An. sacharovi* and *An. maculipennis*, also *Anopheles messeae* and *An. atroparvus* have contributed to the endemism of malaria. Generally *An. messeae* has played a prominent role as a malaria vector in the Danube Valley and Delta, while *An. maculipennis* was mainly responsible for malaria transmission in the Romanian plains and *An. sacharovi* and *An. atroparvus* have been primary vectors at the Black Sea coast (Zotta, 1938; Zotta et al., 1940; Ciuca, 1966). All these issues represent the concept of "malaria stratification", which indicates a good relation between the distribution of the different anophelines species and the great "malaria geographic lines" (Nicolescu, 1996). Moreover a new species of the *An. maculipennis* complex, named *Anopheles daciae*, was recently first described in Romania (Nicolescu et al., 2004). It seems likely that *An. daciae* could be widespread in Eastern Europe and the Balkan States, and also could be responsible for malaria transmission in these regions that is currently attributed to *An. messeae*.

In order of relevance, *An. sacharovi*, *An. superpictus* and *An. maculipennis* were considered the main malaria vectors in Greece (Belios, 1955, 1978). During the recent years several autochthonous cases of *P. falciparum*, *P. malariae* and *P. vivax* have been diagnosed in Northern Greece (Kampen et al., 2002). At respect, it is important to note the proximity of this region to an unstable malaria country as Turkey. In Turkey malaria is still one of the most important vector-borne diseases in Turkey (Kasap et al., 2000; Alten et al., 2003), even remaining some endemic areas with hundreds of vivax cases yearly. The most important vectors in Turkey are *An. sacharovi* and *An. superpictus* (Kuhn et al., 2002), taking *An. maculipennis*, *An. claviger* and *Anopheles hyrcanus* a secondary role in malaria transmission.

Re-Emergence of Malaria and Dengue in Europe 491

semiactive winter habits but not a complete diapause. In conclusion, northern malaria existed in a cold climate by means of summer dormancy of *P. vivax* hypnozoites in addition to the indoor feeding activity of overwintering *Anopheles* females previously mentioned. In other Scandinavian countries such as Sweden or Denmark, besides the anophelines which has been mentioned above, there have been described other potential malaria vectors: *An. atroparvus* and *Anopheles plumbeus* (Ramsdale & Snow, 2000). Although *An. messeae* was probably the main vector during the malaria epidemics in Sweden, some authors proposed that *An. atroparvus* may have maintained malaria endemicity in certain coastal localities in the south of the country (Jaenson et al., 1986). Regarding to *An. plumbeus* there are several aspects that should be pointed to understand the increasing epidemiological importance of the species in Central Europe. *An. plumbeus* is the only hole breeding species of the genus *Anophele*s in Europe. Although it is a strictly dendrolimnic species, during dry periods females can also lay the eggs in small domestic and peridomestic containers, as well as other artificial breeding sites below the ground such as catch basins and septic tanks with water contaminated with organic waste (Bueno Marí & Jiménez Peydró, 2011). There are several reports in Europe about the presence of larvae in a biotope different from the tree cavity (Aitken, 1954; Senevet et al., 1955; Rioux, 1958; Tovornik, 1978; Bueno Marí & Jiménez Peydró, 2010a). Moreover, remarkable populations can also be found in urban situations, where the larvae develop in tree holes in gardens and parks, especially in Central Europe where *An. plumbeus* has increased in numbers during the last decades and can be a major nuisance species (Becker et al., 2010). This is a very important issue, because the continuous development of this species in urban environments could increase considerably the possibilities of interaction between malaria vectors and humans. In fact, *An. plumbeus* has been suspected to be responsible for two recorded cases of locally transmitted malaria in London, United Kingdom (Blacklock, 1921; Shute, 1954) and other two cases recently reported in Duisburg, Germany (Krüger et al., 2001). Of the five *Anopheles* species present in Britain only two, *An. atroparvus* and *An. plumbeus*, have been confirmed as malaria vectors in United Kingdom (James, 1917; Shute, 1954), while *An. messae* and *An. atroparvus* were the vectors involved in vivax epidemics occurred in Germany during the 20th Century

Therefore, it exists in Europe a latitudinal gradient in relation to the distribution of the species of the *An. maculipennis* complex. Without ignoring the possible participation of several species in malaria transmission cycles, the fact is that in Northern Europe (including European Russia) at 68ºN *An. beklemishevi* prevails as vector, being this species replaced by *An. messae* partially at 63ºN and fully about 59ºN. Around 56ºN *An. atroparvus* begins to acquire an important role in disease transmission and already in Mediterranean countries the situation of malaria receptivity is basically governed by *An. atroparvus*, *An. labranchiae* and *An. sacharovi* in Eastern, Central and Western Mediterranean respectively. As was previously pointed, this situation can be locally modified by the presence of other potential vectors widely distributed in Europe such as *An. claviger*, *An. hyrcanus*, *An. maculipennis* or *An. plumbeus*. Of course climate change could drastically modify not only the distribution of European anophelines, but also their phenology and overwintering patterns. However the changes in agricultural practices have a greater effect on the risk of malaria than an elevation in temperature of approximately 2°C (Becker, 2008), which is considered the average increasing temperature in Europe in next 50 years. Hence habitat modification is probably the factor with more influence in possible changes in malaria

(Kirchberg & Mamlok, 1946).

receptivity all over Europe.

If we analyze the VC of European anophelines we can extract several conclusions. In Spain the populations of *An. atroparvus* were deeply studied by several authors basically during the endemic period (Buen de, 1931, 1932; Buen de & Buen de, 1930, 1933; Torres Cañamares, 1934; Olavarria & Hill, 1935; Lozano Morales, 1946; Zulueta de, 1973; Blázquez, 1974). The estimation of VC shows that *An. atroparvus* was an important malaria vector in different wetlands of Spain mainly during summer months. The VC was especially high for *P. vivax* (in August VC=0.7–21.2) which has a shorter sporogonic cycle than *P. falciparum* (in August VC=0.2-5.3). In September VC values were lower for both *P. vivax* (VC=0.2–9.2) and *P. falciparum* (VC=0.04-2.3) and in October VC values were drastically reduced, but still relevant in the case of *P. vivax* (VC *P. vivax*=0.01-2.1 / VC *P. falciparum*=0.00007-0.02) (Bueno Marí & Jiménez Peydró, 2012). These results are similar to others derivates from different entomological researches carried out in Italy more recently. During August 1994 in Tuscany (Grosseto Province) were reported for *An. labranchiae* VC values ranging from 8.3-32.5 for *P. vivax* and 7.3-26 for *P. falciparum* (Romi et al., 1997). However VC was very low in early July, constituting no real risk for malaria transmission (<0.01 for both *P. vivax* and *P. falciparum*). Subsequently during 1998 in the same province but in areas where only natural anopheline breeding sites were reported, the VC of *An. labranchiae* from mid-July through the end of August ranged from 0.96-3.3 for *P. vivax* and 0.8-2.9 for *P. falciparum* (Romi, 1999). In other Mediterranean areas (North of Morocco), VC of *An. labranchiae* for *P. vivax* also showed high values during summer months (in July VC=17.2; in August=34; in September=18.3), while values from April to June were lower ranging from 0.5-3.7 (Faraj et al., 2008). On the other hand the average VC of *An. sacharovi* was found to be 0.22 (VC ranging from 0.63-0.014) in an endemic area of Southeastern Turkey (Tavşanoğlu & Çağlar, 2008). These last low VC values were probably related with very low percentages of human blood meals by anophelines.

Accordingly, although of course all these values of VC are purely theoretical, it is important to note that can be numerically shown that summer (from July to September, but especially in August) is an excellent season for malaria transmission, at least at receptivity level, in Southern Europe.

#### **2.1.1.2 Malaria receptivity in Northern Europe**

Endemic northern malaria reached to 68°N latitude in Europe during the 19th century, where the summer mean temperature only irregularly exceeded 16°C. It is important to note that precisely 16ºC is considered the lower limit needed for sporogony of *P. vivax* (Garnham, 1988). In Finland *Anopheles beklemishevi* has a northern distribution, while the other common species, *An. messeae*, is dominant in the southern part of the country (Gutsevich et al., 1974; Lokki et al., 1979; Kettle, 1995). Both species are known as an important malaria vectors (White, 1978). Despite other potential vectors, such as *An. claviger* and *An. maculipennis* have been observed (Utrio, 1979; Dahl, 1997), it is not possible to define certainly which mosquito species was most important for the malaria transmission in Finland. This is because temperature conditions of Finland, as well as in other northern countries, should have caused that malaria transmission have mainly occurred in indoor conditions due to transmission of sporozoites throughout the winter by semiactive hibernating mosquitoes (Huldén et al., 2005), since it is well known that in warm conditions the overwintering females of *Anopheles* can take several blood meals (Ekblom & Ströman, 1932; Encinas Grandes, 1982). Therefore, the best malaria vectors in Northern Europe will be those anthropophilic and endophagic anophelines which present hibernating females with

If we analyze the VC of European anophelines we can extract several conclusions. In Spain the populations of *An. atroparvus* were deeply studied by several authors basically during the endemic period (Buen de, 1931, 1932; Buen de & Buen de, 1930, 1933; Torres Cañamares, 1934; Olavarria & Hill, 1935; Lozano Morales, 1946; Zulueta de, 1973; Blázquez, 1974). The estimation of VC shows that *An. atroparvus* was an important malaria vector in different wetlands of Spain mainly during summer months. The VC was especially high for *P. vivax* (in August VC=0.7–21.2) which has a shorter sporogonic cycle than *P. falciparum* (in August VC=0.2-5.3). In September VC values were lower for both *P. vivax* (VC=0.2–9.2) and *P. falciparum* (VC=0.04-2.3) and in October VC values were drastically reduced, but still relevant in the case of *P. vivax* (VC *P. vivax*=0.01-2.1 / VC *P. falciparum*=0.00007-0.02) (Bueno Marí & Jiménez Peydró, 2012). These results are similar to others derivates from different entomological researches carried out in Italy more recently. During August 1994 in Tuscany (Grosseto Province) were reported for *An. labranchiae* VC values ranging from 8.3-32.5 for *P. vivax* and 7.3-26 for *P. falciparum* (Romi et al., 1997). However VC was very low in early July, constituting no real risk for malaria transmission (<0.01 for both *P. vivax* and *P. falciparum*). Subsequently during 1998 in the same province but in areas where only natural anopheline breeding sites were reported, the VC of *An. labranchiae* from mid-July through the end of August ranged from 0.96-3.3 for *P. vivax* and 0.8-2.9 for *P. falciparum* (Romi, 1999). In other Mediterranean areas (North of Morocco), VC of *An. labranchiae* for *P. vivax* also showed high values during summer months (in July VC=17.2; in August=34; in September=18.3), while values from April to June were lower ranging from 0.5-3.7 (Faraj et al., 2008). On the other hand the average VC of *An. sacharovi* was found to be 0.22 (VC ranging from 0.63-0.014) in an endemic area of Southeastern Turkey (Tavşanoğlu & Çağlar, 2008). These last low VC values were probably related with very low percentages of human blood meals by

Accordingly, although of course all these values of VC are purely theoretical, it is important to note that can be numerically shown that summer (from July to September, but especially in August) is an excellent season for malaria transmission, at least at receptivity level, in

Endemic northern malaria reached to 68°N latitude in Europe during the 19th century, where the summer mean temperature only irregularly exceeded 16°C. It is important to note that precisely 16ºC is considered the lower limit needed for sporogony of *P. vivax* (Garnham, 1988). In Finland *Anopheles beklemishevi* has a northern distribution, while the other common species, *An. messeae*, is dominant in the southern part of the country (Gutsevich et al., 1974; Lokki et al., 1979; Kettle, 1995). Both species are known as an important malaria vectors (White, 1978). Despite other potential vectors, such as *An. claviger* and *An. maculipennis* have been observed (Utrio, 1979; Dahl, 1997), it is not possible to define certainly which mosquito species was most important for the malaria transmission in Finland. This is because temperature conditions of Finland, as well as in other northern countries, should have caused that malaria transmission have mainly occurred in indoor conditions due to transmission of sporozoites throughout the winter by semiactive hibernating mosquitoes (Huldén et al., 2005), since it is well known that in warm conditions the overwintering females of *Anopheles* can take several blood meals (Ekblom & Ströman, 1932; Encinas Grandes, 1982). Therefore, the best malaria vectors in Northern Europe will be those anthropophilic and endophagic anophelines which present hibernating females with

anophelines.

Southern Europe.

**2.1.1.2 Malaria receptivity in Northern Europe** 

semiactive winter habits but not a complete diapause. In conclusion, northern malaria existed in a cold climate by means of summer dormancy of *P. vivax* hypnozoites in addition to the indoor feeding activity of overwintering *Anopheles* females previously mentioned.

In other Scandinavian countries such as Sweden or Denmark, besides the anophelines which has been mentioned above, there have been described other potential malaria vectors: *An. atroparvus* and *Anopheles plumbeus* (Ramsdale & Snow, 2000). Although *An. messeae* was probably the main vector during the malaria epidemics in Sweden, some authors proposed that *An. atroparvus* may have maintained malaria endemicity in certain coastal localities in the south of the country (Jaenson et al., 1986). Regarding to *An. plumbeus* there are several aspects that should be pointed to understand the increasing epidemiological importance of the species in Central Europe. *An. plumbeus* is the only hole breeding species of the genus *Anophele*s in Europe. Although it is a strictly dendrolimnic species, during dry periods females can also lay the eggs in small domestic and peridomestic containers, as well as other artificial breeding sites below the ground such as catch basins and septic tanks with water contaminated with organic waste (Bueno Marí & Jiménez Peydró, 2011). There are several reports in Europe about the presence of larvae in a biotope different from the tree cavity (Aitken, 1954; Senevet et al., 1955; Rioux, 1958; Tovornik, 1978; Bueno Marí & Jiménez Peydró, 2010a). Moreover, remarkable populations can also be found in urban situations, where the larvae develop in tree holes in gardens and parks, especially in Central Europe where *An. plumbeus* has increased in numbers during the last decades and can be a major nuisance species (Becker et al., 2010). This is a very important issue, because the continuous development of this species in urban environments could increase considerably the possibilities of interaction between malaria vectors and humans. In fact, *An. plumbeus* has been suspected to be responsible for two recorded cases of locally transmitted malaria in London, United Kingdom (Blacklock, 1921; Shute, 1954) and other two cases recently reported in Duisburg, Germany (Krüger et al., 2001). Of the five *Anopheles* species present in Britain only two, *An. atroparvus* and *An. plumbeus*, have been confirmed as malaria vectors in United Kingdom (James, 1917; Shute, 1954), while *An. messae* and *An. atroparvus* were the vectors involved in vivax epidemics occurred in Germany during the 20th Century (Kirchberg & Mamlok, 1946).

Therefore, it exists in Europe a latitudinal gradient in relation to the distribution of the species of the *An. maculipennis* complex. Without ignoring the possible participation of several species in malaria transmission cycles, the fact is that in Northern Europe (including European Russia) at 68ºN *An. beklemishevi* prevails as vector, being this species replaced by *An. messae* partially at 63ºN and fully about 59ºN. Around 56ºN *An. atroparvus* begins to acquire an important role in disease transmission and already in Mediterranean countries the situation of malaria receptivity is basically governed by *An. atroparvus*, *An. labranchiae* and *An. sacharovi* in Eastern, Central and Western Mediterranean respectively. As was previously pointed, this situation can be locally modified by the presence of other potential vectors widely distributed in Europe such as *An. claviger*, *An. hyrcanus*, *An. maculipennis* or *An. plumbeus*. Of course climate change could drastically modify not only the distribution of European anophelines, but also their phenology and overwintering patterns. However the changes in agricultural practices have a greater effect on the risk of malaria than an elevation in temperature of approximately 2°C (Becker, 2008), which is considered the average increasing temperature in Europe in next 50 years. Hence habitat modification is probably the factor with more influence in possible changes in malaria receptivity all over Europe.

Re-Emergence of Malaria and Dengue in Europe 493

Vulnerability is determined by the number of gametocyte carriers (malaria patients) during the suitable period for malaria transmission. If we analyze the data about imported malaria in Europe in recent years we can extract several conclusions. Malaria represents about 77% of tropical diseases imported in Europe (TropNetEurop, 2010). A total of 65.596 cases were reported in Europe between 2000 and 2009 (Table 3). However this number is clearly underestimated, since in last years the number of malaria reporting sites in Europe has increased significantly. Most of these cases are referred to immigrants (48.5%), and *P. falciparum* (81%) was the dominant species in analytic results. A high percentage of malaria cases in immigrants correspond to Visiting Friends and Relatives (VFR). This group of special epidemiological significance refers to those people who, once are established in their host countries, often travel to their origin countries to visit family or friends. Travels that these people can do to their origin countries exponentially increase the chances of disease contracting, since usually these areas are endemic regions and the stay within resident population and their customs is often long and intense (Gascón, 2006). Therefore this is an important collective to promote the need to take appropriate prophylactic measures during travels to endemic areas. Several studies have revealed that only 16% of VFR search for medical advice pre-travel, being malaria prophylaxis practically nonexistent in this collective (Leder et al., 2006). The European countries with higher number of imported malaria cases reported yearly are France and Germany, usually followed by other like Spain, Italy or Belgium. As it was shown before, malaria receptivity is remarkable in concrete

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 Total

7411 (50)

8544 (50)

8904 (52)

9509 (57)

10.143 (59)

65.596

**2.1.3 Vulnerability** 

regions of these countries.

1120 (32)

3313 (38)

4555 (47)

5561 (44)

6536 (47)

*P. falciparum* 78.4% 70.0% 77.6% 82.4% 81.2% 81.6% 87.8% 82.8% 83.9% 84.0% 81% *P. vivax* 11.5% 13.9% 11.7% 10.4% 11.2% 10.2% 7.5% 8.3% 8.2% 8.6% 10.1% *P. ovale* 5.2% 5.3% 3.4% 3.1% 3.5% 4.4% 2.8% 4.3% 3.9% 3.1% 3.9% *P. malariae* 2% 5.9% 4.3% 1.5% 1.5% 1.7% 1% 1.2% 1.5% 2.3% 2.3% Unkn./Coinf. 2.9% 4.9% 3.1% 2.7% 2.4% 2.2% 0.9% 3.4% 2.7% 4.4% 2.7% Imm./Refu. 30.5% 35.4% 44.8% 50.2% 54% 54.6% 52.8% 50.8% 55.2% 56.7% 48.5% For. Vis. 11.8% 14.6% 7.5% 9.1% 7% 9.2% 10.3% 3.3% 5.1% 9.2% 8.7% Eur. E.C. 53.8% 44.4% 38.9% 30.6% 30% 26.1% 26.2% 35.2% 34% 26.4% 34.5% Eur. Exp. 3.9% 5.6% 8.8% 11.1% 9% 10.1% 10.7% 10.7% 5.7% 7.7% 8.3%

*Note 1*: Unkn./Coinf. (Plasmodium species unknown or coinfection of various species), Imm./Refu. (Immigrants/Refugees), For. Vis. (Foreign Visitors), Eur. E.C. (Europeans living in EC), Eur. Exp.

Table 3. Imported malaria in Europe between 2000-2009 (TropNetEurop, 2010).

Cases (sites reporting)

(European Expatriates).
