1

Educational material created within the Erasmus+ Strategic

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Online courses with videos for the field of veterinary communication dealing with

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MALARIA

GUIDE OF MAIN INFECTIOUS DISEASES TRANSMITTED FROM NON-HUMAN

ANIMALS TO HUMANS – MALARIA IN HUMANS AND ANIMALS

This project has been funded with support from the European Commission.

This publication reflects the views only of the author, and the Commission cannot be held

responsible for any use which may be made of the information contained therein.

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Main author: Liviu Miron

Co-authors:

Romania: Dumitru Acatrinei, Olimpia Iacob, Larisa Ivanescu, Lavinia Ciuca , Constantin Roman, Raluca Mindru,

Andrei Lupu, Andrei Cimpan, Gabriela Martinescu, Elena Velescu, Mioara Calipsoana Matei, Doina Carmen

Manciuc, Alina Manole, Doina Azoicai, Florentina-Manuela Miron, Gianina-Ana Massari, Anca Colibaba, Cintia

Colibaba, Stefan Colibaba, Elza Gheorghiu, Andreea Ionel, Irina Gheorghiu, Carmen Antonita, Anais Colibaba

Croatia: Nenad Turk, Zoran Milas, Zeljana Kljecanin Franic

Lithuania: Tomas Karalis, Rūta Karalienė, Virginija Jarulė, Leonora Norviliene, Donata Katinaite, Daiva

Malinauskiene

Italy: Ilaria Pascucci, Ombretta Pediconi, Antonio Giordano

Copyright© 2016-2019 University of Veterinary Medicine Ion Ionescu de la Brad, Iasi (Romania). All rights

reserved.

University of Veterinary Medicine Ion Ionescu de la Brad, Iasi (Romania) is the beneficiary of the Erasmus+

project Online courses with videos for the field of veterinary communication dealing with

prevention, diagnosis and treatment of diseases transferable from animals to humans

2016-1-RO01-KA203-024732

No part of this volume may be copied or transmitted by any means, electronic or mechanical, including

photocopying, without the prior written permission of the 2016-1-RO01-KA203-024732 project partnership.

GUIDE OF MAIN INFECTIOUS DISEASES TRANSMITTED FROM NON-HUMAN ANIMALS TO HUMANS – MALARIA

IN HUMANS AND ANIMALS

Online courses with videos for the field of veterinary communication dealing with prevention, diagnosis and

treatment of diseases transferable from animals to humans

2016-1-RO01-KA203-024732

www.zoeproject.eu

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Project partnership:

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DEFINITION AND HISTORY

DEFINITION Malaria is an important disease caused by parasites from the Plasmodium genus that are

transmitted to humans through the bites of the infected female of Anopheles mosquitoes,

mostly in tropical and subtropical regions. It is the disease with the largest prevalence on

the globe, millions of people being infected annually in Africa, India, South-East Asia, the

Middle East, Central and South America, which means that almost 50% of the world

population is at risk of infestation with this disease.

HISTORY

Since the end of the Pleistocene, people migrating to the north have brought the parasite of

malaria with them. Since the Neolithic Age and the Bronze Age, the skeletal remains

indicate the existence of chronic anemia caused by Plasmodium falciparum infection. The

widespread presence of thalassemia (Mediterranean anemia) - a genetic disease that

provides some protection against malaria, and which indicates a long history of contact with

the malaria pathogen has been reported since ancient Greek and ancient Rome. The

introduction of agriculture around 7000 B.C. resulted in increased population stability and,

consequently, the creation of favorable conditions for the transmission of malaria. The

deforestation that began around this time contributed to the preservation of the necessary

habitat for the development of Anopheles mosquitoes. Around 400 AD (Jesus Christ's birth),

a series of ecological changes occurred, with a gradual warming and drought in the

Mediterranean region and a gradual increase of the sea level.

The first literary mention of the existence of autumn fever was registered in the Iliad by

Homer (800 or 900 BC), when Achilles was fighting with Hector, and king Priam "sees a fatal

sign emblazoned on the heavens, which brings such killing fever down on wretched men".

We cannot know precisely if he refers to malaria, but it is obvious that the devastating fever

is associated with harvesting time, which from an ecological point of view is ideal for the

development of the mosquito’s biological cycle. It is certain that the subsequent texts

confirm that the disease had become significant in ancient Greece. Hippocrates (460-377

BC) was the first to describe in detail the seriousness of tertiary infections associated with

humid areas, noticing even splenomegaly (chronic malaria symptom) as a manifestation

present in people living in swampy areas. The physicians Praxagoras and Heraclites offered

similar descriptions of a disease in Greece. Horace, Lucretius, Martial and Tacitus were

among the Latin authors who mentioned the disease in ancient Rome (Reiter, 2001). The 2nd

century A.D. occasioned a series of Galen’s and Celsus’s writings, which, beside symptoms

and treatment, also mention the existence of three species of parasites - P. falciparum, P.

ovale, and P. vivax, which are frequently present. In the Middle Ages there were mentions

of a period of coldness, so in 763-764 A.D. ice presence is mentioned on the Adriatic Sea.

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Nevertheless, the Visigoths, Ostrogoths, and other barbarian armies were destroyed by

malaria and several popes died from malaria on their journeys to Rome. The decline of

malaria started in the second part of the 19th century. Denmark suffered from devastating

epidemics until the 1860s, especially in the rural areas, and then the transmission lowered,

and even stopped at the beginning of the 20th century. The situation was similar in Sweden,

though isolated cases were reported until 1939; also, in England there was a gradual

decrease until 1880, after which it became rare, except for a short period after World War I.

In Germany the transmission also lowered after the 1880s; the last outburst took place in

Paris in 1865, during the construction of the Grand Boulevards, while in the rest of France

the disease had stopped, as in Switzerland. The decline of malaria in all these countries

cannot be attributed to climatic changes, because it occurred during a phase of temperature

increase. Other factors that have been involved in the decline of malaria include: the

improvement of drainage and the adoption of new agricultural methods which helped

eliminate the mosquito habitats. The demographic changes and people’s living conditions

had a significant role, the populations decreased in the rural area as manual work was

replaced by machines, thus reducing the availability of humans (compared to animals) as

hosts for mosquitoes and of humans as hosts for the parasite. The improvement of medical

services and the use of quinine on a larger scale lowered the rate of survival of the malaria

parasite in the human host.

Fig.1. Malaria endemic areas

(Source: Hay and Snow, 2006)

Malaria is endemic stable in regions where anophelines are anthropophilic and have a

higher rate of survival, the temperature and the humidity are generally higher, and there is

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a relatively small seasonal variation. The disease is hard to control because the transmission

is extremely efficient – most of the people being infected several times a year. The severe

disease and death generally occur in children and immigrants. The temperature plays an

important role in the transmission of the disease because the extrinsic incubation of the

parasite inside the mosquito depends on it. Thus, the vector Anopheles gambiae can be

found both at sea level and at 3000 m above sea level, but endemic malaria disappears at

over 1,800 - 2,000 m (Reiter, 2001).

ETIOLOGY, ETIOLOGIC AGENT, TAXONOMY, MORPHOLOGICAL

DESCRIPTION AND BIOLOGICAL CYCLE

ETIOLOGY Malaria is a disease produced by a protozoan of the genus Plasmodium, which infects the

red blood cells (RBC) through innoculation in humans by the female mosquito of the genus

Anopheles during blood feed (Fig. 2). There are five species of mosquitoes parasitizing

humans, and which can be transmitted from one person to the other: P. falciparum, P.

vivax, P. ovale (two species), and P. malariae. Lately, an increasing number of human

infections has been reported about species P. knowlesi parasitizing the monkey in the forest

regions of South-East Asia, and especially in Borneo Island.

Fig. 2. Intraerythocytic (RBC) infestation

(Source: Boddey and Cowman, 2013)

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TAXONOMY Plasmodium is a protozoan belonging to the Plasmodiidae family, order Haemosporidia and

phylum Apicomplexa, which, together with dinoflagellates and ciliates, form the

superphylum Alveolata belonging to the Eukaryotic kingdom.

MORPHOLOGICAL DESCRIPTION The malaria parasite undergoes four development stages in humans (hepatic schizonts, then

intra-erythrocytic trophozoites, schizonts, and gamonts), and three development stages

inside the mosquito vector (ookinete, oocyst, and sporozoite).

The schizonts are activated under the action of certain factors, transforming into merozoites

which penetrate the erythrocytes, with the following stages:

The signet ring stage: the parasite has the shape of a ring, taking 1/3 of the

erythrocyte total volume. The protoplasm at the other end of the nucleus is thicker,

blue, and the nucleus is red. In the middle, there is a colourless vacuole full of

nuclear liquid. The duration of this phase is approximately 12 hours for Plasmodium

falciparum, varying according to the species.

The deformed ring stage: the ring is deformed at the side opposite the nucleus. The

dark brown granulations that appear represent the melanin pigment resulting from

the deposits of iron in hemoglobin. The duration is 12 hours.

The amoeboid stage I: there are protoplasmic pseudopods in all directions, with

various forms and sizes. The nucleus grows and the vacuole decreases or disappears.

The melanin pigment is abundant in the whole mass of the parasite.

The adult amoeboid stage II: the parasite grows, extending in the whole erythrocyte,

getting a more regular shape. The nucleus is big. The melanin pigment is abundant

and uniformly spread. The duration of both amoeba stages is 16 hours.

The pre-rosette stage: the nucleus starts to divide into 2, 3, 4 and 6 nuclei.

The rosette stage: the nuclei are divided at the maximum (16-24). Sometimes we can

also see the division of the protoplasm surrounding each nucleus, resulting in

merozoites of 40-80μm diameter. They place themselves at the periphery of the

parasite, on several planes, giving the rosette a muriform aspect. The melanin

pigment remains in the middle of the parasite. The duration of pre-rosette and

rosette stage is 8 hours, and the duration of the whole schizogonic cycle is 48 hours.

Sporogonic forms are represented by gametocytes. A hypertrophied, un-deformed

erythrocyte of regular contour has a round or oval-shaped hematozoon, with an intense

blue protoplasm, a small, dense, crimson-red nucleus, no vacuole, and very fine and

uniformly spread granulations of melanin pigment. This is the female gametocyte or

macrogametocyte of 7-14μm diameter. The male gametocyte or microgametocyte has a

reddish-blue protoplasm, a big nucleus with diffuse chromatin, and coarse melanin pigment

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placed in heaps around the nucleus. The microgametocytes of the mosquito are long (15-

25μm length), and they are produced by exflagellation of fertilised round

macrogametocytes to form ookinetes (15-20 x 2-5μm), which migrate through the intestinal

wall to form ovoid oocytes (up to 50μm diameter) on the exterior surface. The oocyst

produces thousands of thin long sporozoites (~ 15μm length) that eventually infect the

salivary glands.

BIOLOGICAL CYCLE The malaria biological cycle is one of the most fascinating and complicated of all organisms,

presenting a special interest, especially in molecular biology, cellular biology, and

immunology. The malaria vector transmission by mosquitoes of the Anopheles genus was

established by Grassi at the end of last century. The mosquitoes involved in the transmission

of malaria are: A. hyrcanus, A. labranchiae, A. maculipennis maculipennis, A. maculipennis

atroparvus, A. maculipennis messae, A. plumbeus, A. claviger, A. sacharovi. In our country,

the following species were signalled: A. maculipennis, A. messae, A. atroparvus, A.

sacharovi, and Anopheles labranchiae.

Malaria infection in humans involves an asymptomatic stage in the liver and a symptomatic

erythrocytic stage. Drug research has focused solely on the erythrocytic stage, which has a

limited potential for transmission blocking and prophylaxis. A new technique may be used

for the study of drugs with regard to their efficacy on the hepatic stage using the parasites

expressing luciferase, i.e., bioluminescence. Most of these studies concern the activity of

the drugs during the erythrocytic stages, since there are few known drugs to actually

destroy the hepatic phases. The best known and efficient drug is primaquine, the only drug

used also for the treatment of the hypnozoite forms of Plasmodium vivax, which may lead

to relapses after months, even years from the infection. The problem is that primaquine

produces haemolytic anaemia in individuals with glucozo-6-phosphate (G6PD) deficit, such

enzyme deficit being specific mostly in regions affected by malaria from Africa, South

America and Asia (Adoubryn et al, 2004; Derbyshire, 2012).

THE SPOROGONIC CYCLE OF THE MALARIA PARASITE Changing the habitat causes significant losses for the malaria parasite, leading to an

unbalance in population density. This unbalance is compensated by the growth and

reproduction in cellular niches protected by the host’s immune responses (Frevert, 2004). In

the stage of ookinete and sporozoite, the parasite undergoes the greatest losses, these

stages taking place in the mosquito vector (Silvie et al, 2008). The ookinete develops from

the zygote in the lumen of the host’s middle intestine, being the result of fertilisation

between a female macrogametocyte and a male microgametocyte. The ookinete crosses the

layer of intestine epithelial cells on the apical side, reaching eventually the basal lamina. As

a response to the invasion, the body activates its protection mechanisms, leading to a

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decrease in the parasite population density. The ookinetes are transformed into oocysts,

which constitute an extracellular development stage, leading to the formation of

sporozoites which are released in the cavity of the mosquito body, thus invading afterwards

the salivary glands (Al-Olayan et al, 2002; Vlachou et al, 2006) (Fig.3). During this migration,

there is a decrease in parasite population density, the sporozoites innoculated to the

mammal host reaching the liver in a very short time. In 1948, Shortt and Garnham

discovered that the sporozoites infect the hepatocytes, developing a stage there before

infecting the erythrocytes. The hepatic stage is asymptomatic, certainly interesting from a

scientific point of view as regards vaccine production. Here is the amplification up to 10,000

times of the number of parasites, culminating with the release of exo-erythrocytic

merozoites. The merozoites invade the erythrocytes, consequently initiating the

erythrocytic cycle, which is pathogenic (Vaughan et al, 2008). Today we know the expression

of genes and proteins involved in the evolution of the stages inside the mosquito and the

pre-erythrocytic stage (Greenwood et al, 2005; Kappe et al, 2001; Lasonder, 2008). The

discovery of the gene responsible for the hepatic stage led to the formation of attenuated

sporozoites, obtaining an attenuated live vaccine.

The parasite sexually reproduces only once in the mosquito body. The factors initiating the

formation of gametocytes are not very well known. The ingestion of gametocytes during the

blood feed activates the formation of gametes (gametogenesis) in the mosquito’s intestinal

lumen.

A derived molecule of xanthurenic acid, together with the change in temperature and pH,

may start the gametogenesis of the male gender under the form of exflagellation (Billker et

al, 1997; Billker et al, 1998). The protein P48\45 is essential in enabling the male gamete to

fertilise the female gametes (which have the protein P47expressed on the surface).

Fertilisation takes place to form the zygote, which after meiosis is transformed into an

ookinete that crosses several epithelial cells of the intestine (Mueller et al, 2005). The

ookinetes cross the epithelial cells up to the basal lamina and transform into oocysts, which

grow extensively, and in 10-14 days lead to the production of thousands of sporozoites.

After migrating through the haemolymph, the sporozoites resulted from oocysts attach to

the basal side of the acinar cells of the salivary gland. The sporozoites cross the acinar cells

and enter the ducts of the salivary glands where they are transmitted to the mammal host

during the blood intake (Pimenta et al, 1994; Robert and Boudin, 2003).

9

Fig. 3. The sporogonic cycle of the malaria parasite

(Source: Personal collection)

THE ERYTHROCYTIC SCHIZOGONIC CYCLE OF THE MALARIA

PARASITE The mammal host is infected with sporozoites from the salivary glands of mosquitoes.

Approximately 100 sporozoites are injected by a single mosquito (Medica and Sinnis, 2005).

They remain in the derma for a period of time, displaying an apparently random movement

until they find a blood vessel, and then migrate to the liver (Mota et al, 2001; Amino, 2007).

After injection, the sporozoites remain in the derma for 1-3 hours and then migrate to the

liver (Yamauchi, 2007). Not all the sporozoites get into the circulation and into the liver,

some of them are stuck at the injection place to be probably be eliminated by phagocytosis.

Some of them reach the lymphatic system, that is, the lymphatic ganglions, where the

response of T and CD8 cells is activated. These cells are capable of eliminating the

developing parasites from the liver (Chakravarty et al, 2007; Amino et al, 2008). In the skin,

the sporozoites actively migrate through the cells, leading to perturbation of the plasmatic

membrane of the host cell. For the cellular crossing, at least three proteins are involved:

SPECT-1, SPECT-2, and phospholipase. The sporozoites lacking SPECT-1, SPECT-2 are

immobilised in the derma as a result of the impairment of the cell crossing capacity (Amino

et al, 2006; Sturm et al, 2006; Ménard, 2007; Sturm et al, 2006; Aly et al, 2009). After

sporozoite innoculation by Anopheles vector, the exo-erythrocytic cycle starts, with a

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duration, according to the species, as follows: Plasmodium falciparum – 6 days; Plasmodium

vivax – 8 days; Plasmodium ovale – 9 days; Plasmodium malariae – 15 days. Some

sporozoites of Plasmodium vivax and Plasmodium ovale, when penetrating the hepatocytes,

remain in a latent stage for a long period of time (a few years), being called hyposonts

(Cogswell, 1992).

Fig.4. Asexual replication cycle of the parasite inside the red blood cells

(Source: Silvie et al, 2008)

(1) The extracellular merozoites are randomly attached to the surface of the erythrocytes.

(2) Then, the apical end of merozoites is directed towards the surface of the erythrocytes.

(3) Formation of Tight junction and the active merozoites enter the erythrocytes. (4)

Invasion of merozoites occurs by simultaneous formation of a parasite phorous vacuole.

After the invasion, the merozoites become ring-shaped. (5) This stage is characterised by a

big digestive vacuole, where digestion of hemoglobin results in the formation of the malaria

pigment known as hemozoin. (6) The parasite grows intraerythrocytic, and it is called

trophozoite. The hemozoin pigment accumulates in the digestive vacuole. (7) DNA

replication precedes the future cell, and the process is called schizogony. (8) The merozoites

secrete exoneme, which initiates the exit from the parasite phorous vacuole and red blood

cells (RBC) of the host. (9) The exiting merozoites adhere to the adjacent erythrocytes in a

few seconds. (1) Initiation of a new erythrocytic cycle (Fig. 4, 5).

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Fig.5. Various erythrocyte stages (1-2 ring form, the mature schizont 3, 4-gametocytes) of Plasmodium vivax,

Giemsa stained smear x 1000 (Source: Personal collection)

Sporogonic forms: gametocytes (Fig. 6). A hypertrophied un-deformed erythrocyte of

regular contour has a round or oval-shaped hematozoon, with an intense blue protoplasm, a

small, dense, crimson-red nucleus, no vacuole, and very fine and uniformly spread

granulations of melanin pigment. This is the female gametocyte or macrogametocyte of 7-

14μm diameter. The male gametocyte or microgametocyte has a reddish-blue protoplasm, a

big nucleus with diffuse chromatin, and coarse melanin pigment placed in heaps around the

nucleus (Field and Shute, 1956; Gratzer and Dluzewski, 1993; Moore et al, 2008).

Innoculated in humans, the parasite undergoes a series of transformations, first in the liver

(this constitutes the exo-erythrocytic or tissue cycle), then it penetrates the RBC, where it

develops until it leads to the destruction of RBC (the erythrocytic or asexual cycle). The

parasite released from the destroyed cells parasitizes other cells again (Tolle, 2009; Sturm et

al, 2006) (Fig. 7).

a b

Fig.6. a. Mature schizonts and merozoites

Col. Giemsa x100, immersion objective,

(Source: Personal collection)

b. Stage of gametocytes (macrogametocit pink,

blue microgametocit)

Col. Giemsa x100

(Source: Personal collection)

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The mosquito innoculates the parasite under the form of sporozoites (infecting

forms for humans) which form, upon reaching the liver, a big plasmodial mass from which

merozoites come off, reach the blood and penetrate the RBC. Inside the RBC, the parasite

goes through the amoeboid stage, then the rosette stage, and then it destroys the RBC

releasing the merozoites which will parasitize other RBC where the cycle starts over again

(Hafalla et al, 2011). Gametocytes (males, females) also result from the erythrocytic cycle,

joining in the body of the mosquito and forming zygotes. In the intestinal lumen, the zygotes

transform into ookinetes, from which the sporozoites are produced after a series of

transformations. Located in the salivary glands, they are innoculated in humans (Vlachou,

2006).

Fig.7. Life-cycle of Plasmodium-vivax

(Source: http://www.microbiologynotes.com/life-cycle-of-plasmodium-vivax/)

The Anopheles females biting a sick person whose blood contains gametocytes and

schizonts ingest with the blood a variable number of parasites under these two forms. The

schizonts are digested in the stomach of the mosquitoes, and the gametocytes ensure the

continuation of the sexual or sporogonic cycle, which leads to the sporozoites. It has been

demonstrated that some mosquitoes contain two genes which encode the

carboxypeptidaze from the middle intestine, which are involved in the parasite

development with Anopheles gambiae (Lavazec and Bourgouin, 2008; Aly et al, 2009).

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EPIDEMIOLOGY

MALARIA TRANSMISSION By vectors – the vector transmitted to humans, and at the same time the compulsory hosts

for the sexual stage of the parasite, is represented by the mosquitoes of the Anopheles

genus (of the approximately 400 species on the globe, only 30-40 can be malaria vectors in

natural conditions). The period of formation of sporozoites in the salivary glands varies

between 7 and 30 days, according to the parasite species and environmental temperature.

By blood transfusion - malaria post-transfusion known from 1911 appears from latent

asymptomatic malaria carriers. In most cases, post-transfusion transmission was done by P.

malariae (which gives persistent forms with long time blood carriers), and more rarely by P.

vivax and P. falciparum.

The period of human contagiousness is the time that the infesting gametocytes of the

malaria parasite persist in the blood of the sick person, and it varies with Plasmodium

species. Consequently, the gametocytes of P. malariae can persist in the blood even up to

53 years, those of P. vivax for 1-3 years, and P. falciparum up to 1 year. The Anopheles

female remains an infesting agent its entire life.

There are three possible modalities of malaria transmission from one sick person or

plasmodia carrier to healthy people: in natural conditions via Anopheles mosquito bites;

intrauterine, from a sick mother to the foetus through placenta or during childbirth; by

transfusion - by blood containing the pathogen during medical manipulation or injections in

aseptic conditions with contaminated needles.

Malaria transmission occurs in the mosquito activity season, depending on the transmission

intensity and duration, and on the influence of natural and social factors with long term

effect.

MALARIA VECTORS In the majority of cases, malaria is transmitted through the bite of the mosquito female of

the Anopheles genus. There are more than 400 different species of mosquitoes of the

Anopheles genus; around 30 species represent vectors of major importance for malaria (Fig.

8). The intensity of the transmission depends on the factors relating to the parasite, vector,

human host and the environment. Each mosquito species of the Anopheles genus has its

own aquatic habitat; for example, some prefer small fresh water sources, such as ponds and

the water accumulated in the traces of hooves, which are abundant in tropical countries

during the rainy season.

Understanding the biology and behaviour of the Anopheles mosquitoes may help us

understand the way in which malaria is transmitted and also elaborate appropriate control

14

strategies. The factors that affect the capacity of a mosquito to transmit malaria include its

innate susceptibility with regard to Plasmodium, choosing the host and its longevity. The

factors that should be taken into account when elaborating a control program include the

malaria vectors’ susceptibility to insecticides and the preferred feeding and resting place of

adult mosquitoes. Transmission is more intense in places where the lifetime of mosquitoes

is longer (the parasite has enough time to finish its development within the mosquitoes) and

mosquitoes prefer feeding off humans, not animals. The long lifetime and the

anthropophilic character of the African mosquito species represents the main reason for

which almost 90% of malaria cases in the world are in Africa. Transmission depends as well

on the climatic conditions that may affect the number and survival of mosquitoes: rainfalls,

temperature and humidity. In many places, transmission is seasonal, reaching its peak

during and after the rainy season. Malaria epidemics may break out when the climate and

other conditions favour a sudden transmission in areas where people have low or no

immunity against malaria. They may also appear when people with low immunity travel to

areas with intense malaria transmission, for example, to find a workplace as refugees.

Fig.8 Global distribution of dominant or

potentially important malaria vectors (Robinson Projection)

(Source: Kiszewksi et al, 2004)

Mosquitoes are spread all over the world with the exception of the arctic area and from the

approximately 3500 existing species, three quarters come from tropical and subtropical

15

areas. The medical importance of these insects is represented by their need to feed on

blood in order to obtain the necessary protein for egg maturation. The feeding is done with

the help of a complex salivary secretion that facilitates the transmission of some pathogenic

agents to the mammal host while sucking the blood. The ecology, behaviour, survival of

mosquitoes and the dynamics of the transmission of diseases are strongly influenced by

climatic factors: temperature, rainfalls, humidity, wind, and sunshine duration. Also, the

development time of the pathogenic agent in the salivary glands of mosquitoes is directly

influenced by temperature (Reiter, 2001).

Malaria is transmitted through the bite of the female mosquitoes of the Anophleles genus.

In Europe, the mosquitoes of the Anopheles maculipennis complex have been incriminated

as vectors for the disease. The first person who noted as early as 1926 the existence of a

complex of the maculipennis species was Falleroni, who described the morphological

differences regarding the chorion of these species. There are approximately 430 species

registered as belonging to the Anopheles genus, out of which approximately 60 species are

considered vectors of malaria (Reiter, 2001).

The complexes of species are very closely related groups of species that are hard to

differentiate from a morphological point of view. Anophles maculipennis Meigen, the

historical vector of malaria in Europe and the Middle East, was the first complex with twin

species discovered in mosquitoes (Falleroni, 1926; van Thiel, 1927). The search included

morphological studies based on eggs (Falleroni, 1926; Falleroni, 1932; Corradetti, 1934; de

Buck and Swellengrebel, 1934; Hackett and Lewis, 1935; Korvenkontio et al, 1979), on

chromosomes (Frizzi, 1952; Frizzi, 1953; Kitzmille, 1967; Stegnii, 1976; Stegnii and Kabanova,

1976), sequences/DNJ (Marinucci et al, 1999; Proft et al, 1999; Linton et al, 2001; Linton et

al, 2002; Linton et al, 2003; Sedaghat, 2003; Porter, 1991), ecology (van Thiel, 1927; de

Buck and Swellengrebel, 1934; Hackett and Missiroli, 1935), hybridization (de Buck and

Swellengrebel, 1934; Kitzmiller et al, 1967), larvae (Bates, 1939; Buonomini, 1940; Suzzoni-

Blatger and Sevin, 1981; Boccolini et al, 1986; Suzzoni-Blatger et al, 1990; Romi, 2000), and

on wing characteristics (de Buck et al, 1933; Ungureanu and Shute, 1947; Sedaghat et al,

2003). As a result of all these studies the presence of eight species was established within

the Palearctic maculipennis complex:

A. atroparvus van Thiel, A. beklemishevi Stegnii and Kabanova, A. labranchiae Falleroni, A.

maculipennis, A. martinius Hingarev, A. melanoon Hackett, A. messeae Falleroniand, A.

sacharovi Favre (White, 1978; de Zulueta et al, 1983; Cianchi et al, 1987; Ribeiro et al, 1988;

Linton et al, 2002; Sedaghat et al, 2003; Knight and Stone, 1977; Kettle, 1995) (Fig. 9).

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Fig.9. Species of the Palearctic maculipennis complex

(Source: https://ecdc.europa.eu/en/disease-vectors/facts/mosquito-factsheets/anopheles-sacharovi;

http://mosquito-taxonomic-inventory.info/file/5212)

All the members of this complex are considered vectors of malaria in Europe. Taking into

account the medical importance of the maculipennis complex, the differences regarding the

vectorial capacity and species distribution that are thought to be influenced worldwide by

global warming, what becomes necessary is a sure method of identifying the species. Once

the Anopheles female becomes infected with Plasmodium, it will have sporozoites in its

salivary glands for the duration of its entire life, therefore infecting a new host every time it

feeds on blood. The vectorial capacity is an index of the transmission capacity of the malaria

vector which is influenced by the density of the vector (by climatic conditions: temperature

and humidity; by geographic conditions: seasonal variations, and by zoophilic or

anthropophilic preference (Mullen and Durden, 2002; Marquardt, 2004; Toole, 2009). In the

absence of the preferred host, feeding behaviour may be modified (Boete, 2006; Pages et al,

2007; Nitzulescu and Gherman, 1990). A. atroparvus, A. labranchiae and A. sacharovi have

been incriminated as main vectors of malaria in the region of Europe (Vicente et al, 2011).

The duration of the gonotrophic cycle (the distance between egg laying) is of approximately

2 days, and the duration of the evolution of sporozoites is approximately 12 days, so that at

least 6 gonotrophic cycles are needed before the female becomes infected. The duration of

the sporogonic stage varies depending on the Plasmodium species involved; as such, for P.

falciparum, between the ingestion of gametocides and the presence of sporozoites in the

salivary glands, 12 days are needed at a temperature of 25°C, and 23 days, respectively, at

20°C. Below 18°C and over 33°C the cycle stops (Bruce-Chwatt and Zulueta, 1980). The

global distribution of malaria depends a lot on the intrinsic characteristics of the vector, the

vector’s competence (the capacity of an Anopheles species to ensure the complete

development of the parasite) and its vectorial capacity (Pratt and Moore, 1993). The

A. atroparvus (van Thiel,1927)

A. beklemishevi (Stegnii and Kabanova, 1976)

A. labranchiae (Falleroni,1926)

A. maculipennis (Meigen, 1818)

A. messae (Falleroni, 1926)

A. martinus Hingarev

A.sacharovi (Favre, 1903)

Anopheles melanoon Hackett, 1934

17

incapacity of the malaria parasite to develop in some mosquito species is tied to the

absence of some metabolic factors that are essential for the development of the parasite or

the presence of some toxins that inhibit the development of the parasite. The presence of

the vectors largely depends on the local conditions (Mouchet et al, 1999). The climatic

changes represented by an increase in temperature by as little as 0.5°C may lead to an

increase in the mosquito population by 30-100% (Patz and Olson, 2006). This highlights the

necessity of permanently reporting the Anopheles species that are present and the density

of the populations in order to be able to quantify the control measures for malaria. In the

mountainous areas of Africa, the incidence of malaria has increased since 1970 in direct

correlation with the change in climatic factors, more precisely the fact that an increase in

temparature has been recorded (Pascual et al, 2006). Molecular identification of the

Maculipennis complex species is important if we consider its medical signifocance, the

vectorial capacity and the distribution of the species that seems to be directly influenced by

global warming (Sedaghat et al, 2003). Anopheles claviger has a large distribution, all the

way from the sub-Carpathian areas to the lowlands. The larvae nests are usually found in

copses, orchards, in places shaded from direct sunshine and with moderate temperatures.

The larvae live even during winter, under the ice. It is a fairly aggressive mosquito which

attacks humans even during the day.

Anopheles hyrcanus is a widely spread species in the Danube Delta, but also in the

countryside, near brackish water. Its spreading epicenter is in the Middle East, where it is a

malaria vector, and it steadily decreases as we move towards the West.

Anopheles maculipennis, Anopheles messeae, Anopheles sacharovi are part of the

maculipennis group, which comprises anopheles members with stained wings, these dark

stains originating in the agglomeration of scales on some areas of the wing veins.

The species of the Maculipennis group is characterized by a high plasticity of the larvae,

which adapt to fairly varied larvae nests. The safest criterion for differentiating the three

anopheles species from the Maculipennis group is the egg. For each species, the ventral part

of the egg, directed upwards while floating on water, is characteristically ornated.

Anopheles maculipennis is a widely spread species in the country, near forests and on river

valleys. The well oxygenated freshwaters provide the egg-laying place, with a lower average

temperature, spared from direct sunlight. The female is zoophile and occasionally

anthropofile. During the winter hibernation is complete, but with a gonotrophic

concordance, in shelters away from humans, in open stables or hollows exposed to the cold.

Anopheles messeae is spread all over the low plains in the country and in places with

stagnant waters (ponds, lakes, the lower third part of rivers that are usually floodable, the

floodable area of the Danube).

18

The larvae nests are represented by stagnant, open freshwaters, with a shallow bottom and

abundant vegetation, warm and well exposed to the sunlight. The larvae develop well

especially on the banks of these waters and at the periphery of the islands of vegetation.

The female is predominantly zoophile but it also feeds off humans. It has a complete

hibernation, with gonotrophic concordance, in shelters fairly exposed to the cold.

Anopheles atroparvus is spread in the entire country, in areas with more or less salty waters.

In this regard, the larvae have a great plasticity of adaptability, from larvae nests in

freshwater to saltwaters (4-5%). They are not very sensitive to lower temperatures

(Ungureanu and Shute, 1947).

The female is over wintering in intradomestic and peridomestic shelters, free from the cold,

where it can find food. It does not hibernate completely and it awakens from time to time to

bite humans or cows, but it is predominantly anthropophile.

The Anopheles female in general mates once and keeps the sperm in the spermathecal,

from where it is used during the entire life of the female for fertilizing the eggs. For the egg

maturation an intake of blood is needed, the female being able to travel up to 3 km in

search of a host, generally attracted by the carbon dioxide emanated by the host

(Smallegange RC et al, 2005). It seems that the development of the gametocytes (the

infected form for the human) is more prosperous within the Anopheles gambiae species,

which is the main malaria vector in Africa (Lacroix R et al, 2005). Following mating, 48 hours

are needed for egg maturation, then they are laid on water, and their properties differ

depending on the Anopheles species (size, sunlight exposure, type of natural or artificial

water source, unpolluted). Adult females lay 50-200 eggs per oviposition. Eggs are laid

directly on water and may float on both sides. They are not resistant to drying and hatching

in 2-3 days, even though the sampling may take up to 2-3 weeks under lower temperature

conditions. These characteristics are valid for the Anopheles species from the rural areas

and the periphery of the urban area, the risk of malaria transmission being higher in the

rural area (Pages F et al, 2007). The preference for feeding inside (endophage) or outside

(exophage), for an ambiental environment – endophile or exophile, varies from one species

to the next or within the same species, depending on the geographic area. These

characteristics play an important role in the development of fighting strategies against the

anopheles (Pages F et al, 2007). The females feed from dusk till dawn, the peak of attack

differs depending on the species, A. cruzii, A. bellator from South America being active

during daytime. The flight of the species of the Anopheles genus is silent and the bite is not

painful.

The Anopheles genus comprises species of high medical importance, some 500 species

overall, of which 50 are malaria vectors (Harbach, 1994).The adults are characterized

morphologically by palps equally long as the trunk in both sexes, simple scutellum and

19

abdomen without scales. The hypopygium is made up of forcipules without lobes, claspers

are present, paraprocts do not chitinize, and the phalosome is often ornated with apical

growths. The larvae are missing the siphon. The eggs are laid isolatedly on the surface of the

water and have a pair of lateral floats, more developed in the species growing in freshwater

areas and smaller in the species from saltwater areas. The development cycle from egg to

adult takes from 8 days (at a temperature of 31°C) to 20 days (Foster and Walker, 2009).The

development period of the malaria agent inside the mosquito (extrinsic incubation) varies

between 10 and 21 days, depending on the Plasmodium species. The life span of a mosquito

in nature may be calculated exactly by estimating that over 20% of the mosquitoes would

survive more than a period of extrinsic incubation of 14 days. Thus, it can be concluded that

it is not the density of the mosquito population that is important, but rather their longevity,

therefore the purpose is to shorten the life of mosquitoes by using insecticides.

ANOPHELES MACULIPENNIS

FEMALES

The colour is dark or medium brown, even though colour and size variety is wide. The

characteristic that differentiates all the other European Anopheles species are the wings

with dark scales that form characteristic stains. The trunk has a dark brown colour and the

maxillary palps are the same colour and length as the trunk. The vertex has a long whitish

scale-patch directed towards the front. The scutum has a grey band that narrows towards

the front. The lateral parts of the scutum are brown in the front and dark brown on the rear

side. The scutum is brown with narrow golden scales. The postnotum is light brown and the

pleura are dark brown. The femur is dark brown on the dorsal part and light brown on the

ventral side, the tibia is brown and of a lighter colour towards the tip, the tarsus is dark

brown. The wings have dark, unevenly distributed scales that form black stains close to RS

and R2 3 and M. In A. sacharovi there are dark stains on the wing veins and on the rear

edge. The abdomen is brown or dark brown with a golden brown stripe (Capinera, 2002).

MALES

In the culicidae species of the Anopheles genus, or at least in the ones present in our

country, the coxit has missing lobes. The base of the coxit lengthens laterally, with three

apophyses or apodemes. The external and dorsal internal apodeme serve for attachment on

segment 9. The basal apodeme or basal plate, on which the penial and anal muscles are

inserted, reaches segments 9 and 8, and on the other side it is articulated with the

parameres of segment 10.

LARVAE

Quite variable in pigmentation and size, the larvae from the northern part of Europe are

usually bigger, with a closed head capsule, the antenna is almost straight, slightly spiculated,

almost 2/3 of the head size. The front needle (5-C to 7-C) is long. The needle trowns on the

20

abdominal segments I and II are rudimentary, but well developed on segments III-VII. In the

anopheles species there are bristles that ramify, the same as on the head and thorax, as

opposed to the culicinae whose bristles ramify from the base.

For the Anopheles species, a few other characteristic structures may be also observed on

each abdominal segment:

a chitinous tergal plate whose size varies depending on the species;

accessory plates, posterior to the median plate, one, two or three on each

segment;

needles grown on each segment, representing a surface-active floating system;

these bristles may be used to differentiate the species of the Maculipennis group

from the Anopheles hyrcanus species.

The 8th abdominal segment, having the same form as the other 7, constitutes the support

for some structures of taxonomic importance. On the lateral walls of segment 8 are situated

the abdominal comb or abdominal spines, one on each side. In the anopheles species, the

siphon is missing and the spiracles (stigmatic orifices) are held by a spiracular plate

(Cosoroaba, 1992; Cosoroaba, 2000).

ANOPHELES LABRANCHIAE (FELLERONI, 1926) The larvae develop in stagnant water, fresh or saltwater, in the coastal areas of Europe,

preferring the heat. Hibernation is done in the dark by the adult female, in stables or natural

cavities. The diapause may be complete or incomplete, with occasional blood feasts.

Females are mainly anthropophage, occasionally feeding off domestic animals.

Anopheles labranchiae is an endophilic species, living in households, stables, animal shelters

and only occasionally outdoors, in natural shelters (tree hollows). The presence of larvae has

been recorded from April to October. The flight of this species is limited to 2-5 km (Senevet

and Andarelli, 1956). Anopheles labranchiae has a limited distribution in the South and

South-East of Europe. It was reported in the South-East of Spain, Corsica, the coastal areas

of Italy, Sardinia, Sicily. In Northern Africa, the species is found in Morocco, Algeria and

Tunisia (Zahar, 1990). Anopheles labranchiae has registered an increase in numbers in

Sardinia in the last 35 years. Between 1940 and 1953, a period in which malaria was

eradicated, Anopheles labranchiae numbers dropped significantly, the eradication campaign

consisting of insecticides (DDT). The expansion of urban areas and changes in agricultural

practices have led to a new increase in Anopheles labranchiae populations.Sardinia presents

a biological interest due to its central geographic position (lat. 38-41 N, long. 8-9E) to the

West of the Mediterranean, halfway between Africa and Europe. Sardinia is the second big

island in the Mediterranean (250 x 120 km). The reproduction places for Anopheles

lambrachiae are varied: swamps, rivers covered with vegetation, holes, channels, basins,

sunny areas preferably. Anopheles labranchiae and Anopheles sacharovi are considered to

be the most efficient vectors in the transmission of malaria in the Palearctic region (Marchi,

21

1987). The main reproduction sites for Anopheles labranchiae in this region remain the rice

fields, a fact that continues to be a main concern for agriculture.

In Corsica, Anopheles labranchiae continues to be the main malaria vector, together with

Anophles sacharovi. Other species are considered possible vectors elsewhere in Europe,

namely Anopheles algeriensis, Anopheles atroparvus, Anopheles claviger, Anopheles

hyrcanus, Anopheles maculipennis SS, Anopheles marteri, Anopheles melanoon, Anopheles

messae, Anopheles petragnani, Anopheles plumbeus, Anopheles superpictus, Anopheles

subalpinus; because of their physiologic characteristics, especially zoophilism, their part is

minimal in the transmission of malaria (Toty C et al, 2010).

The center of Italy used to be a hyperendemic area for malaria up until the mid-20th century,

when the malaria eradication campaign drastically reduced the number of vectors

incriminated for malaria transmission, Anophele labranchiae.The introduction of rice crops

led to a substantial increase of this species, causing concerns for the re-emergence of

malaria in this area.

The changes in the use of land, the climatic variations, and the seasonal demographic

variations have all contributed to the growth of the Anopheles labranchiae populations, thus

increasing the risk of local malaria (Boccolini et al, 2012).

In Morocco, Anopheles labranchiae is considered the main vector of malaria, the last case of

local malaria being recorded in 2004. Malaria control in this region includes the use of the

biological control method, namely, the mosquitofish (Gambusia holbrooki). Insecticides are

used as a last resort, but they still occupy an important place in the National Plan for

Malaria Control (NMCP). The insecticide used for destroying the Anopheles labranchiae

larvae, also recommended by the WHO, is an organophosphorus insecticide called

temephos, 0.125mg / L being the smallest concentration with a 100% mortality rate (Faraj,

2010). Temephos is the insecticide used to destroy the larvae and DDT is used in the control

of adult mosquitoes. Large scale use of insecticides in agriculture may constitute a problem

since Anopheles labranchiae is frequently found on agricultural land. Anopheles labranchiae

is the only species of the maculipennis complex found in North Africa. Anopheles

labranchiae is the main vector in the transmission of malaria in Italy as well.

In the region of Tuscany there was a case of local malaria registered in 1997 and, based on

an in-depth study, it was noted that Anopheles labranchiae was the only Anopheles species

identified in the region. A further study of the same species proved that the probability of

malaria transmission is high in August, when the period needed for the development of the

parasite inside the mosquito is reached, namely, 11 days for Plasmodium falciparum and 10

days for Plasmodium vivax, at a temperature of 25°C, the daily average temperature (Romi,

1999). An article published in 1998 noted that Anopheles labranchiae had disappeared in

22

Spain (Eritja et al, 1998). The reason behind the lack of similarity between the distribution of

Anophles atroparvus and Anophles labranchiae seems to be the temperature, the latter

preferring warm waters. Both species prefer brackish waters and lagoons, but Anopheles

labranchiae prefers to lay eggs in freshwaters. In Sardinia, larvae have been found in almost

all habitats, with the exception of shaded ones (Sinka et al, 2010). The A. labranchiae

females attack humans readily; Romi et al. have signalled the presence of human blood in

86% - 90.7% fed females (Romi et al, 1997). Anopheles labranchiae may be both endo and

exophile, depending on the available habitats (D’Alessandro et al, 1971). Similar to A.

atroparvus, A. labranchiae proved to be refractory to infections with African strains of P.

falciparum, even though the Center for Production and Infection of Anopheles (Centre de

Production et d'Infection des Anophèles - CEPIA) from Paris has positively demonstfrated

the infection of A. labranchiae with an African strain of P.falciparum NF54 (Toty C et al,

2010).

ANOPHELES ATROPARVUS (VAN THIEL, 1927) Anopheles atroparvus becomes active at temperatures starting from 15°C, when it may

constitute a risk for the transmission of malaria (Knottnerus, 2002). For reproduction, it

prefers brackish waters or freshwaters, well-lighted areas (Potugalia, Spania). It prefers

relatively cold waters, and the larvae have been found in marshes, ditches, flooded basins in

the ground, pools formed in riverbeds, tanks for transporting cement, rice fields, fountains

and basins, as well as in water collected in used tyres. It prefers sunny sites with filamentous

green algae and other aquatic plants. Anopheles atroparvus is a predominantly zoophile

species, preferring in order: rabbits, horses, cows, swine and sheep, as such being the main

vector of myxomatosis in rabbits. However, it has been described as anthropophile as well,

since it can be found in households and has been recorded feeding off humans. Hibernation

is done in stables or households, the adult female feeding periodically, without laying eggs.

This has led to the spread of malaria in the Netherlands, Great Britain and other parts of

Europe at the beginning of the 20th century. The duration of the diapause depends on the

length of the day and the temperature; therefore, it varies with altitude and may vary from

September to October in Northern Europe, or from November to February in Southern

Europe. It is generally a littoral species which may be found in the South-East of Portugal,

along the coasts of the Atlantic Ocean, Great Britain and the Mediterranean Sea. In South

and South-East Europe it has an uneven distribution. In Macedonia it is largely spread in the

lowland areas, visibily dominant only in the areas with saline/alkaline soils from the

Pannonian Plain. In Portugal it is the most common and widely distributed species (Becker

et al, 2003).

In a 2010 study which aimed to identify the most frequent vectors from 49 countries in

Europe and the Middle East, comprising 2891 locations, Anopheles atroparvus was signalled

in the highest number of locations - 1044 (Sinka et al, 2010). Anopheles atroparvus was the

main incriminated vector in the transmission of malaria in the British Isles, being the main

23

source of P. vivax. In the case of P. falciparum it has not been established yet if the parasite

is capable to reach the end of its lifecycle and transmit the infection. Most research notes

that Anopheles atroparvus is capable of transmitting tropical strains of Plasmodium

falciparum, although it may transmit European strains as well (Cambournac, 1994). A study

by Sousa mentions the existence of an Anopheles atroparvus in Portugal which is capable of

transmitting tropical strains of P. falciparum (Sousa, 2008). Anopheles atroparvus is largely

spread in England, Scotland, Wales, and Ireland, together with Anopheles messeae, from

which it cannot be differentiated morphologically, unless it is in its egg phase (Snow, 1998).

Anopheles atroparvus is largely spread in the countries from the Mediterranean basin, but

because of its zoophilic character, it does not present a major risk in malaria transmission,

unless the populations show high density (Eritja et al, 1998). Its distribution varies in Europe,

it is absent in some Mediterranean regions, such as the South of Italy, Greece and Turkey

(Vicente et al, 2011). Anopheles atroparvus, together with Anopheles messae, are

considered the main vectors of malaria in the UK. The genetic identification based on the

ITS2 sequence was confirmed by Linton et al. in 2002. A study carried out in 2001 on the

river banks of the Rhine and Meuse, in the south-western part of the Netherlands,

demonstrated that the populations of Anopheles atroparvus, which is considered the main

and only vector of malaria in the area, had decreased a lot, with only 4 out of 150 larvae

examined representing A. atroparvus, and the rest being identified by PCR as A. messeae

(Takken, 2002).

ANOPHELES SACHAROVI (FAVRE 1903)

This species is the easiest to distinguish of the entire Maculipennis complex by the light

colour of the mesonot and the median band on the scutum, which is absent in the other

members of the complex. The lateral parts of the scutum are brown-yellow, the same as the

middle part. The stains on the wings are not very obvious, actually barely perceptible in

older individuals of the species, compared with the other members of the complex. A.

sacharovi eggs are distinguished by the missing floats and the larvae are smaller than those

of other members of the complex (Becker et al, 2003).

Anopheles sacharovi is considered to be the most efficient malaria vector, a main vector in

Turkey, Siria, the Middle East, as well as in Southern Europe (Yaghoobi-Ershadi et al, 2001;

Romi et al, 2002; Sedaghat et al, 2003).

Anopheles sacharovi is very flexible in choosing its habitats both as an adult and as a larva,

and it is found in small water sources with vegetation. It prefers habitats with freshawater,

but it has been described as the most tolerant species in the entire maculipennis group, up

to a salinity of 20%. It may survive in waters of up to 38-40°C, in stagnant waters, also being

able to withstand a weak current (Pener and Kitron, 1985). This species prefers sunny sites,

marshes being a perfect habitat, but it has also been found on the borders of rivers,

streams, springs, pools and ditches.

24

As a result of eradication campaigns and of using DDT in the past, a zoophilic characteristic

was initially recorded, but an anthropophilic behaviour is currently noted (Sinka et al, 2010).

Unlike the other members of the Maculipennis complex, A. sacharovi attacks throughout

the day, with maximum intensity in the early evening between 20:00-22:00 (Alten et al,

2003).

Hibernation is incomplete, the female feeds multiple times without laying eggs (Manguin et

al, 2008; Kasap, 1995). Anopheles sacharovi is an important vector in Turkey, Siria, Israel,

Jordan and the Middle East (Zahar, 1990; Sedaghat et al, 2003). For reproduction, it prefers

small water sources with aquatic vegetation. Anopheles sacharovi is considered the main

vector in the Balkans, but in Romania it was declared extinct after the eradication of malaria

in this area (Zahar, 1990), and it is very rarely found in Italy (because of environmental

changes and changes in agricultural practices). The species is still abundant in Turkey and

North-East Greece (Romi, 1999). Anopheles sacharovi is a species that quickly gains

resistance to insecticides, as was shown by a study carried out in 2012. The levels of

irritability described by the WHO are DDT 4%, dieldrin 0,4%, malathion 5%, fenitrothion 1%,

permethrin 0,75%, and 0,05% for deltamethrin. The analysis results of insecticide doses for

this species signalled resistance to DDT, tolerance to dieldrin and some sensitivity to

fenitrothion, malathion, permethrin, and deltamethrin. DDT had the most irritating effect,

while deltamethrin was the least irritating (Vatandoost and Abai, 2012).

In the laboratory, under varied ecological conditions, at altitudes ranging from 353 m to

1126 m in the Sanliurfa Turkish province, Anopheles sacharovi did not present any

significant difference from egg stage to adult stage, or any developmental differences.

Anopheles sacharovi is the main vector of malaria in Turkey and it is, together with

Anopheles superpictus and Anopheles pulcherrimus, the most important vector of malaria in

the former Soviet Union, even though A. messeae was directly involved in the re-emergence

of malaria in Russia and Ukraine. Anopheles maculipennis was considered the main malaria

vector on the Caspian Sea coast and Anopheles sacharovi is considered the main malaria

vector in the central plateau of the country. The members of this complex of species are

active in Northern Iran from May to September, with a peak in July, developing well in rice

fields and around clean waters, with adults found in animal shelters in proportion of 95%.

Blood feeding usually starts at 19.00, with a peak between 20.00-21.00, and the ELISA

immunoenzymatic test shows a preference for cattle, followed, to a smaller degree, by

sheep and poultry. The anthropophilic index for this species in the Northern Iran is 1.7-4.9%

(Djadid et al, 2007). Azerbaijan and Armenia have been free from malaria for more than 30

years, up until 1994, when it re-emerged with A. sacharovi and A. superpictus as vectors.

ANOPHELES MESSEAE (FALLERONI, 1926) Anopheles messeae is the most widely spread member of the maculipennis complex,

distribution ranging from Ireland in Europe to Asia, China and Rusia. For A. messeae, a series

25

of genetic polymorphisms has been identified, and five different haplotypes have been

defined relative to the different geographic areas of its distribution; it has not been possible

to demonstrate if these polymorphisms are indicative of an altered behaviour in these areas

(Di Luca et al, 2004). The larvae are found in shaded waters, with a very smooth flow or that

are stagnant, such as the borders of lakes and marshes. The larvae have been found in sites

with reed, floating aquatic weeds, algae, such as small lakes and dunes. It is a species found

predominantly in the floodable areas of great rivers, such as the Danube, the Rhone, the

Sava or the Rhine. It is very rarely or almost never found along the coast and in

mountainous areas (Becker et al, 2003). Anopheles messeae has been found in human

households, in indoor spaces, staircases, basements, stables, outdoor spaces (hollows,

underground spaces, ponds), but very few have actually been collected (Sinka et al, 2010).

In the Netherlands, A. messeae is not considered a risk for the transmission of malaria due

to its zoophilic characteristics, that is, feeding off humans only if populations are too dense

and animals too few (Takken et al, 2002).

Unlike A. labranchiae and A. atroparvus, A. messeae enters diapause and hibernates, the

adult female prefers abandoned buildings without animals, not needing to feed on blood

until spring and getting its energy from the fat reserves (Jaenson et al, 1991).

Anopheles messeae may transmit malaria at low temperatures, as low as 4 °C, in which case

44 days are needed for the mosquito to become infectious. This is how the cases in Scotland

and Norway can be explained. In Great Britain, Anopheles messeae and Anopheles

atroparvus are responsible for the malaria cases signalled there (Knottnerus, 2002).

Anopheles messae is rarely found in waters that contain more than 1.5g NaCl/l. The species

prefers shaded areas, being similar to A. typicus and A. melanoon (Zahar, 1990).

Anopheles messeae is an important malaria vector in Western Asia, and it was recently

identified as a main vector for the re-emergence of malaria in Ukraine and Russia (Sedaghat

et al, 2003).

ANOPHELES MELANOON (HACKETT 1934) Anopheles melanoon is found in freswaters and in rice fields from Northern Italy. Also, it is

found in marshlands, in large surface stagnant waters, on the borders of rivers and lakes,

ponds and pools. In Sardinia the larvae may be found in freshwaters, sunny places in spring

and shaded areas in summer. They were also reported in rice fields (Becker et al, 2003).

Hibernation is done with full diapause, and the species is mainly zoophile, feeding off

humans occasionally, both outdoors and indoors. It was identified in the Anatolian region in

Turkey, where malaria is endemic, together with A. sacharovi and A. maculipennis (Akiner

and Cağlar, 2010). A. melanoon is a rare species with limited distribution in South-West and

southern Europe.

26

ANOPHELES MACULIPENNIS (MEIGEN, 1818) Anopheles maculipennis has its reproduction habitat in cold waters, in mountainous areas,

but it has also been found close to the sea, in association with Anopheles messeae in

running waters. Also, it was found in warm waters, for example in Naples, Italy (Zahar,

1990).

Anopheles maculipennis has been identified as a major malaria vector on the Caspian

seacoast in Iran. The characteristic reproduction sites are the undisturbed areas of river

courses and banks, rice fields and artificial basins. In the mountainous regions from Central

Europe the species may be found at altitudes of 1000 m and more, being the only member

of the complex found under this type of conditions. Altitudes of 2190 m and up to 2,300 m

are reported by Bulgaria and Turkey. Hibernation is complete but may be short in areas with

a warm climate. Females are generally zoophile, feeding mainly off bovines but also off

swine and poultry, and when these are in small numbers, they may feed off humans as well,

both outdoors and indoors. The species is predominantly endophile, using stables and

houses as resting sites.

Distribution: A. maculipennis S.S. is largely spread in the entire Europe. Except for the

southern tip of the Iberian Peninsula, it is recorded in almost every European country. It

extends towards the East and the South-West of Asia and the Persian Gulf. It is considered

to be a more continental species, with humidity needs considerably smaller than those of A.

messae and A. atroparvus (Becker et al, 2003).

ANOPHELES DACIAE It was signalled by Nicolescu et al. in 2004 as a new species of the Maculipennis complex.

Captured on the Black Sea coast and in the plains adjacent to the Danube, in the south of

Romania (Nicolescu et al, 2004), this species is similar to Anopheles messae (the difference

was established based on the ITS2 sequence, noting a difference of 1.03% bases). Anopheles

daciae was signalled in the UK as well, proving for two years consecutively that it is a stable

population, with no susceptibility of artefact (Linton et al, 2005).

ANOPHELES SUBALPINUS (HACKETT ET LEWIS 1935) Anopheles subalpinus reproduces in freshwaters or slightly saline waters, such as sunny

swamps and ponds with vegetation, avoiding shadowed places for reproduction. It is also

present in the rice fields, preferring stagnant waters with horizontal vegetation in spring. In

summer the eggs are easier to recognize, as with A. typicus and A. messae, while during

spring and winter they are black (Zahar, 1990). The regular reproduction sites can be found

up to 1,200 m high. Hibernation in adult females is complete, usually in caves and

abandoned buildings. A. subalpinus is considered a strongly zoophilic species that rarely

feeds off humans. Females are endophile and may be found in large numbers during the day

27

in stables and barns, and more rarely in households or buildings. It is a South-European

species with an interval between the Iberian Peninsula, up north to the Mediterranean

countries, and the lowlands area around the Caspian Sea (Becker et al, 2003).

ANOPHELES GAMBIAE It is considered the most important malaria vector on the continent of Africa (Fig. 10). In

1960, the existence of an Anopheles gambiae complex or Anopheles gambiae sensu lato was

recognised, which comprises the following species: Anopheles arabiensis, Anopheles

bwambae, Anopheles melas, Anopheles merus, Anopheles quadriannulatus, Anopheles

gambiae sensu stricto, Anopheles coluzzii, Anopheles amharicus. Taken individually, the

species of the complex are very difficult to differentiate morphologically, this being possible

to some extent based on the larva or the adult female.

Fig.10. Anopheles gambiae

(Source: https://cameronwebb.wordpress.com/tag/anopheles-gambiae/)

However, the behavioural traits differ; for example, Anopheles quadriannulatus is a

saltwater and mineral water species, A. melas and A. merus are saltwater species, while the

rest of the species are freshwater species. Anopheles quadriannulatus is zoophile by

preference, feeding off animals, while Anopheles gambiae sensu stricto generally feeds off

humans, being therefore considered anthropofile. Identification through molecular methods

remains the most certain method, and the suggestions put forth by Fanello’s team carry

enough weight and have had important implications for the subsequent control measures

(Fanello et al, 2002). The members of the Anopheles gambiae complex are spread in tropical

Africa, the South of the Sahara Desert, with Anopheles arabiens presence extending to the

28

South of Arabia. Anopheles gambiae s.s. is distributed in Sub-Saharan Africa, including the

Madagascar. In open tree-less habitats, higher temperatures are recorded at noon

compared to wooded habitats, so that the anthropic effect shortens the gonotrophic cycle

of Anopheles gambiae female by 2.6 days (52%) and by 2.9 days (21%) during the dry

seasons and the rainy seasons respectively, as compared to the wooded areas (Patz and

Olson, 2006).

Adult females may be distinguished from a morphological point of view, the muscles around

the mouth lengthening, actrually as long as the trunk, and, while in static position, the

abdomen is kept in the air (Foster and Walker, 2009). The colour varies from light brown to

grey, with pale stains of yellow, white or cream colour and dark stains on the wings. The size

of adults is relatively small, varying between 2.8 and 4.4 mm (Gillies and de Meillon, 1968).

The eggs measure approximately 0.47 to 0.48 mm, are convex on the dorsal side and

concave on the ventral side, also presenting a polygonal drawing. They are laid individually

on water, each egg having floats on both sides and a low resistance to drought (Foster and

Walker, 2009).

The Anopheles gambiae larvae usually live in freshwater areas, with fresh, deep and

temporary sources of water, such as soil depressions, puddles and pools. This characteristic

may help avoid predators because the larvae can develop very quickly (six days from egg to

adult under optimal conditions); the vegetation is reduced as a result of the temporary

nature of the habitats; floating algae, rice fileds and sites lacking vegetation may constitute

appropriate habitats as well. Anopheles gambiae usually reproduces in places populated by

humans; after feeding, the female prefers to rest on the walls near or inside houses. Such

behaviour may offer an opportunity to eradicate the species from African villages and

houses by using residual insecticides. Females find their hosts using a variety of sensory

receptors which react to movement, carbon dioxide gradients and sweating (Konate et al

1999; Meijerink et al, 2000; Roberts and Janovy, 2000).

THE EPIDEMIOLOGY OF MALARIA IN HUMANS

THE SOURCE OF THE PATHOGENIC AGENT

Humans are the most important source of pathogens, with the exception of Plasmodium

malariae, which is common to humans, African monkeys and some species of South

American monkeys. Non-human primates are naturally infected with different species of the

parasite, some of them related to human species (P. cynomolgi, P. simium, P. brasilianum, P.

knowlesi). These parasites should be closely monitored because there have been cases of

29

laboratory infestations (experimental) with P. cynomolgi and natural infestations

(transmitted via mosquito from animal to human host) with P. knowlesi and P. brasilianum,

a fact that made possible documenting these species as a source of pathogens for human

malaria (Heymann, 2012; Bocsan et al, 1999).

Humans can be sources of pathogens in three situations: when they are ill, pre-infected

carriers (the future ill people during the incubation period) or chronic postinfected carriers.

The incubation period differs depending on the species (10-12 days for malaria induced by

P. falciparum, 13-15 days when P. vivax and P. ovale are involved, and 27-42 days for

malaria cases induced by P. malariae). The periods may be prolonged (8-10 months) if

chemoprophylaxis schedules are not correctly used. Also, the contagious period (during

which gametocytes are in the blood) varies depending on Plasmodium species and

treatment response: the chronic carrier status can be prolonged to 1-2 years in untreated P.

falciparum infections, up to 4 years for the infection with P. vivax and P. ovale, and up to 30

years or the entire life for the P. malariae infection (Heymann, 2012; Bocsan et al, 1999).

Another important source of pathogenic agents is represented by Anopheles mosquitoes.

MODES AND WAYS OF TRANSMISSION

Of the over 400 species of Anopheles, about 80-85 can transmit malaria and only 45 are

considered vectors of major importance (Bocsan et al, 1999; Dondorp et al, 2017; Al-Eryani

et al, 2016). In the mosquito's body, after going through the sporogonic cycle, gametocytes

are converted to sporozoites which will be innoculated through the bite from the

mosquito's salivary glands directly to the receptive human host. The sporogonic cycle has a

variable duration depending on the Plasmodium species (for P. vivax - 10-14 days, for P.

falciparum - 16-23 days and for P. malariae - 15-30 days) and the ambient temperature

(Bocsan et al, 1999).

Malaria can be transmitted either directly or indirectly. The direct mode can be effected

through the mosquito bite, by contaminated blood transfusion or medullary transplant.

Also, malaria can be transmitted from mother to foetus in case of placental abnormalities

(congenital malaria). The indirect mode involves the use of contaminated transmission

routes, generally contaminated needles and syringes for intravenous drug users.

Dissemination of the pathogen occurs as long as the asexual forms can be found in the

circulating blood of the source. In the body of the receptive subject the merozoites continue

to develop in the peripheral blood with their own periodicity, leading to malarial access. For

this category there is no relapse because there are no hypnozoites, but they become

infective to the mosquito due to the existence of gametocytes. The most common pathogen

involved in transfusion malaria is P. malariae (which persists asymptomatically for a long

period of time), and the most severe clinical form of the disease is produced by P.

falciparum (Bocsan et al, 1999; Luca, 2002).

30

Malaria transmission does not occur at an ambient temperature below 16°C or above 33°C,

nor at an altitude of more than 2000 meters above sea level because the development of

the sporozoite cannot take place (Dondorp et al, 2017).

As a result, the behaviour of the vector and the way the hematophagy lunch is performed

have an important role in the transmission of the disease, most often through a bite done in

the evening, during the night or in the early morning. Another major factor in the

transmission of malaria is that a gametocyte population occurs in the human host after a

series of asexual cycles for P. falciparum and somewhat earlier for P. vivax. The density of

gametocytes is higher among patients with high parasite density, among

immunosuppressed individuals (children), and when antimalarials to which the parasite is

resistant are used. Some genetic hemoglobinopathies (examples: siklemia, G6PD deficiency

or thalassemia) confer protection against malaria produced by P. falciparum (Dondorp et al,

2017; Abdul-Ghani et al, 2016).

RECEPTIVITY/ SUSCEPTIBILITY

Receptivity is general, with some peculiarities related to resistance to malaria produced by

some types of Plasmodium (Bocsan et al, 1999).

The immune response developed after the natural infection is of the humoral and cellular

type, humoral immunity (antibodies against the asexual red blood cells) being the most

important element of protection. The antibodies appear early during the parasitemia and

reach a maximum level with the decrease in the number of circulating parasites.

In malaria endemic areas, passive natural immunity (IgG of maternal origin) provides

protection for the newborn until around the age of 3 months, after which the level of these

antibodies gradually decreases until total disappearance. In the first year of life of these

children, the immune system is continuously exposed to Plasmodium and the body begins

its own production of antibodies (IgM and IgG) in the presence of parasitemia.

Epidemiological studies show that this immune response reduces the risk of severe

complications and premature death in children who have lived their first year in endemic

areas compared to those outside endemic areas (Bocsan et al, 1999; Luca, 2002).

As concerns the adult population in endemic areas, the appearance of some easier clinical

forms has been observed due to occult immunization through repeated contact with the

mosquito (source of pathogen) over the years (Heymann, 2012).

Studies have shown that a significant percentage of indigenous populations in West Africa

has a natural resistance to P. vivax infection due to the absence of the erythrocyte Duffy

antigen. Heterozygous individuals who sufferer from siklemia are relatively protected

against P. falciparum severe forms of infection, with parasitemia being low, but the

31

homozygous are at increased risk of developing serious forms of the disease, often

progressing to death (Heymann, 2012).

In non-immunized individuals (visitors from malaria-free areas), the disease is always

manifest and most of the time takes on serious forms, and immunity after experiencing the

disease is transient and non-specific (Bocsan et al, 1999).

A special category of receptors is represented by HIV-positive people in endemic areas who

develop the disease more frequently, but often have a low response to anti-malarial therapy

(Heymann, 2012). Receptivity to the disease is also influenced by the normal functioning of

the spleen, which has a role in defending the body (in immunity), but also acts as a filter for

infected erythrocytes (spherical and less deformable). People with functional or anatomic

asplenia develop forms that are rapidly progressive, with much of the circulating cells in the

peripheral blood being infected. Relapses are due to sleeping forms for malaria with P. vivax

and P. ovale, while for the infection with P. malariae persistence of the parasite in the blood

for long periods of time is involved (Bocsan et al, 1999).

MANIFESTATION FORMS OF MALARIA IN THE POPULATION

Malaria can be continuous, seasonal, and intermittent. Seasonal malaria is characterised by

a wave of infection in spring, and a wave of higher amplitude in autumn. Malaria is seasonal

in regions where climatic conditions differ from one season to the other, allowing for the

periodic development of the malaria parasite and vector because the indigenous population

does not have the time to develop sufficient immunity, and consequently the groups of

people at risk of contracting the disease are difficult to define, and mortality can be higher.

In most cases, the epidemics are related to the absence, decrease, or loss of immunity,

which should be alarming for our country, where the population lacks immunity completely.

The parasites develop in the mosquito at a speed that varies with the environmental

temperature (at the Tropics, the parasite gets into the salivary glands and is ready to infect

in approximately 12 days). On the other hand, in the case of stable malaria, transmission is

continuous or regulated per season, and all are either infected or re-infected. According to

the World Health Organisation there are five types of malaria outbreaks (Table I).

Table I. Types of malaria outbreaks according to WHO classification (Source: Colofiţchi, 2007)

1. Inactive residual

natural outbreak

Outbreak where transmission of the pathogen was stopped by

maintaining the morbidity from previous infections

2. Active residual natural

outbreak

Outbreak where antimalaria measures led to a considerable

decrease in the transmission of the pathogen, but transmission

is still performed.

3. Potential new natural

outbreak

Drained outbreak with the presence of cases of malaria

4. Active new natural

outbreak

Outbreak where transmission of the pathogen was performed

from an imported case (it may become endemic)

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5. Drained outbreak Outbreak where transmission of the pathogen was stopped in

a time equal to or higher than three epidemic seasons

GEOGRAPHICAL DISTRIBUTION The report regarding global malaria presented by the WHO shows that for the year 2017 the

situation is alarming since less than half of the countries with ongoing malaria transmission

have managed to achieve the objectives regarding the reduction of the mortality rate

caused by the infection with the Plasmodium parasite. In 2016 there were 216 million

malaria cases at global level, 5 million more compared to 2015. Even though there was a

decrease of malaria cases starting with 2010, the rates stagnated or the trend was

ascending beginning with 2014 in some regions where an increase in malaria cases was

recorded (WHO World Malaria Report, 2017).

It is estimated that in sub-Saharan Africa the proportion of those infected with different

Plasmodium species decreased from 17% in 2010 to 13% in 2015 (CI = 11-15%). The number

of people infected with the parasite dropped from 131 million (CI = 126-136 million) in 2010

to 114 million in 2015 (CI = 99-130 million) (WHO World Malaria Report, 2016). For an

important part of sub-Saharan Africa the transmission is moderate to increased, with a

prevalence of malaria produced by P. falciparum of over 50% (corresponding to a number of

infectious mosquito bites = 10-1000 / year, also called entomological innoculation rate.

Outside Africa, malaria with P. falciparum has a low endemicity with a prevalence rate of

less than 10% (corresponding to an entomological innoculation rate = less than 1 / year). P.

vivax is very common in Asia, where it is responsible for about 50% of malaria cases, as well

as in Central and South America, North Africa and the Middle East. This species of the

parasite is very rare in West Africa, where cases of the P. ovale disease (circulating more

frequently in Asia) prevail. The prevalence of malaria with P. malaria and P. ovale generally

has a lower frequency when compared to other species (Dondorp et al, 2017).

In 2015, 212 million new cases of malaria were estimated worldwide (CI = 148-304 million).

Most of the cases were recorded in the WHO African Region (90%), followed by the WHO

Southeast Asia Region (7%) and the WHO Mediterranean Region (2%). The incidence rate of

malaria declined by 41% globally between 2000 and 2015, and by 21% between 2010 and

2015.

Most of the deaths produced by malaria occurred in the WHO African Region (92%),

followed by the WHO Southeast Asia Region (6%), and the WHO Mediterranean Region

(2%). Most deaths occurred as a result of infection with P. falciparum (99%). It was

estimated that P. vivax was responsible for 3,100 deaths in 2015 (CI = 1800-4900), while

86% were identified outside the African continent (WHO World Malaria Report, 2016).

International travellers could be at risk of malaria infection in 91 countries around the

world, mainly in Africa, Asia and the Americas (Fig. 11).

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But the data is not 100% accurate, exact statistics of the actual situation concerning malaria

in Africa are hard to obtain from the health system, while many treatments and deaths are

not registered.

Fig.11. Malaria Endemic countries

(Source: http://wwwnc.cdc.gov/travel/yellowbook/2012/chapter-3-infectious-diseases-related-to-

travel/malaria.htm)

In Europe, malaria has been eradicated, with the exception of Azerbaijan, Georgia,

Kyrgyzstan, Tajikistan and Turkey. It is estimated that 25-30 million people travel every year

from Europe in areas with ongoing malaria transmission. For the year 2010, WHO reported

6,244 malaria cases in Europe, a smaller number compared to 2000, when 14,703 cases

were reported; in fact, the real figures were estimated to be six times higher. Recent data

from Great Britain and Europe show an important increase in malaria cases, the same

picture being present in USA, where an increase of 14% was registered compared to 2010

(Askling et al, 2012).

In 2011, 40 cases of infection with Plasmodium vivax were reported in Greece, in patients

with no history of travel in a malaria-endemic area, people living in five different districts,

namely Lakonia (n = 34), Attiki (n = 2), Evoia (n = 2), Viotia (n = 1), and Larissa (n = 1). A

group of cases was identified in the Evrotas area, Lakonia, where 27 cases were reported in

Greek citizens who lived in the area and in 7 immigrants from non-endemic countries. Also,

23 cases of P. vivax from Lakonia were reported among immigrant agricultural workers

coming from malaria-endemic countries.

34

On 22nd June 2012 the Hellenic Center for Disease Prevention and Control (KEELPNO)

reported the first indigenous case of malaria caused by Plasmodium vivax in Marathon,

Attica region, Greece. This was a man aged 78 with symptoms since 7th June, with no history

of recent travel in malaria-endemic countries. The Marathon area in Attica was identified as

the possible place of infection. A second case of local malaria caused by Plasmodium vivax

was reportred by KEELPNO on the 17th July. That case was a woman aged 48, a resident of

Evrotas municipality in Lakonia. The patient had reported the beginning of symptoms on the

29th June. She patient had not travelled to a malaria-endemic place in the previous 5 years.

The two indigenous cases of malaria caused by Plasmodium vivax reported in 2012 were

from regions that had been affected in 2011. Currently, there are seasonal (spring-autumn)

measures set in place for preventing malaria, and the risk of spreading the disease to other

countries does not exist because the affected areas are agricultural, not touristic (ECDC,

2012).

Local malaria cases were reported in Germany as well (Krüger et al 2001).

More than 5 million migrants from Africa live in Europe, one third coming from Sub-Saharan

Africa. As a result of the 2011malaria outbreak in Greece, the European Center for Diseases

Prevention and Control (ECDC) organized a conference in January 2012 in order to establish

the risk of transmission of the Plasmodium vivax parasite in Europe. In some member states

of the EU, malaria had been eradicated since 1975 even though the vectors, mosquitoes

from the Anopheles genus, are still present. In the last 10 years sporadic transmission of

malaria was signalled in many countries from Europe: Bulgaria, France, Germany, Greece,

Italy and Spain. Between May 21st and December 5th 2011, 63 cases of malaria caused by

Plasmodium vivax were reported in Greece. Based on the available information, it seems

that the transmission of malaria in Greece could be a result of the reintroduction of the

parasite annually by immigrants. The increase in the number of indigenous cases reported in

2011 indicates that conditions may be favourable for locally sustained transmission in the

affected areas.

The majority of malaria cases in Europe are represented by migrants living in Europe who

visited their friends and relatives (VFR) in their countries of origin, and who were more

vulnerable than tourists. In Paris, a study carried out in a hospital located in a

neighbourhood with an African community presented 239 cases of imported malaria. 81.2%

were cases of VFR that were not treated with prophylactic medication before going to their

native country (mainly from Sub-Saharan Africa, Africa and Comoros) (Develoux et al, 2012).

The data presented by TropNetEurop until 1999 showed that the majority of malaria cases

with Pl. falciparum were imported from West Africa and, as a result, Europeans developed

many complications (Jelinek et al, 2002).

35

Malaria represented a major health problem in France, especially in Camargue, where cases

kept being reported until the beginning of the 20th century. Malaria was eradiacted in

France in 1943 as a result of marshland drainage, animal breeding, construction

development, appropriate use of quinine. Recent entomological studies have signalled

significant mosquito populations from the Anopheles genus, the main potential vector for

the Anopheles hyrcanus malaria based on anthropophilism and their abundance.

With increased international travel, the number of imported malaria cases has increased a

lot starting from 1970 to an average of approximately 6,400 cases per year over the last ten

years in France (Poncon et al, 2008). The number of imported malaria cases has increased in

Europe too, a fact associated with the increase in the number of immigrants relocated in

non-endemic countries who are frequently visiting relatives in their countries of origin. In

the case of immigrants, the clinical symptoms are mild, asymptomatic or delayed, due to the

semi-immunity built after long periods of exposure to stable malaria.

The cases of malaria among immigrants, as well as the asymptomatic cases with microscopic

parasitaemia, lead to an increased risk of transmission and re-emergence of malaria in some

areas in which the vectors are present and the climate conditions are favourable. Malaria

could re-emerge through immigrants and through methods that do not involve the presence

of the vector: blood transfusions, organ transplant, and congenital transmission. Malaria

screening must be carried out among the immigrants who recently arrived from malaria-

endemic countries with the purpose of reducing the risk of clinical malaria, as well as to

prevent local transmission of malaria in the areas in which it had been eradicated.

In 2010 there were 47.300.000 people of foreign origin present in the European Union (EU)

corresponding to 9.4% of the total population. The majority of them, 31.4 million, were born

in countries outside the EU, while 16 million were born in another member state of the EU.

The data regarding those originating from malaria-endemic countries is insufficient.

Estimates indicate the fact that more than 5 million African immigrants could be living in

Europe. Among these, approximately two thirds are from North Africa (Algeria, Morocco

and Tunisia), and the rest are from Sub-Saharan Africa (SSA), with the majority from West

Africa (Ghana, Nigeria and Senegal). Approximately 4 million originate in South-East Asia

and almost 2.2 million in Latin America. The majority of cases were imported in Western

Europe, France, the United Kingdom (UK), Germany and Italy, representing more than 70%

of all the cases (Monge-Maillo and López-Vélez, 2012). The persons infected with the

malaria agent present fever and chills, similar symptoms to those given by the flu.

Untreated, the disease may lead to severe complications and in some cases even death. The

symptoms of malaria appear after a period of incubation of 7 days or longer, depending on

the species of Plasmodium. The fever that occurs with travellers within three months from

exposure represents a medical emergency that must be immediately investigated. Human

immunity is another important factor, especially among adults, in areas with moderate or

36

intense transmission conditions. Partial immunity develops during the years of repeated

exposure to the malaria parasite and even though it never ensures complete protection, it

reduces the risk of a severe form of infection. For this reason, the majority of deaths caused

by malaria in Africa happen with small children, while all age groups are at risk in the areas

with more reduced transmission and low immunity.

In Romania, malaria was eradicated in 1965, and in 1967 the WHO proclaimed Romania a

malaria-free country. The risk of malaria re-emergence in Romania is represented by the

presence of the Anopheles vector, which is part of the Anopheles maculipennis complex and

the pathogenic agent in nature, and by the modification of climate factors (Ivănescu et al,

2016).

According to the European Center for Disease Prevention and Control (ECDC) 2015 report,

the situation of malaria cases in Romania shows an upward trend in recent years. For the

period 2004-2015 "Victor Babes" Hospital for Tropical Diseases reported 150 cases of

malaria in Romania. Of these, 99% come from the African continent and only one case from

Europe – Greece, reported in 2011. Plasmodium falciparum was registered in 75 cases,

followed by P. vivax, P. malariae and P. ovale with the lowest ratio. In 2010 there were 19

cases, in 2011 - 40 cases, in 2012 - 32 diseases, in 2013 - 43, and in 2014 - 57 cases of illness.

In 2015, 30 confirmed cases of malaria were reported (representing an incidence of 0.15 per

100,000 inhabitants, 37% less than in 2014), of which 3 deaths. All cases had the source of

pathogenic agent on the African continent, especially in Equatorial Guinea (6 cases) and

Nigeria (6 cases), and Plasmodium falciparum was predominant (27 cases). Out of the total

number of cases diagnosed, 88% occurred on the continent of Africa, Asia following closely

behind with 8% of the cases (ECDC, 2016; CNSCBT, 2016) (Fig. 12).

Fig.12. Number of malaria cases in Romania

(Source: National Institute of Public Health.

National Center for Communicable Disease Surveillance and Control, 2016)

37

The malaria cases recorded in Romania were all imported as a result of increased travel (for

professional or tourist purposes) to malaria-endemic areas, but also because preventive

measures (nonspecific - mechanical protection or specific - chemoprophylaxis) are not

applied, are unknown or simply ignored by travellers (CNSCBT, 2016).

ELIMINATING AND ERADICATING MALARIA After two failed attempts to eliminate and subsequently eradicate malaria worldwide (1950

and 1960), there is now a new wave of enthusiasm about these goals (Dondorp et al, 2017).

Elimination is defined as the interruption of local transmission of the parasite in a particular

geographic region as a result of sustained efforts by specialists. This stage requires

continued implementation of the necessary measures to prevent re-transmission (WHO -

Malaria, 2018; Bocsan et al, 1999; Velarde-Rodriguez, 2015).

Eradication of malaria represents the disappearance of all cases of the disease (typical or

atypical, manifest or subclinical) caused by Plasmodium, but also the end of the pathogen

circulation as a result of the sustinuted efforts of specialists. Once this goal is achieved,

intervention measures are no longer needed (WHO - Malaria, 2018; Bocsan et al, 1999).

All the countries declaring zero disease cases for three consecutive years are eligible to

apply to the World Health Organization (WHO) and obtain malaria elimination certification.

In the last years, 7 countries have achieved the disease elimination target: United Arab

Emirates (2007), Morocco (2010), Turkmenistan (2010), Armenia (2011), Maldives (2015),

Sri Lanka (2016) and Kyrgyzstan). Between 2000 and 2015, 17 countries eliminated malaria

(WHO - Malaria, 2018; Velarde-Rodriguez et al, 2015).

The WHO “Global Technical Strategy for Malaria 2016-2030” – adopted by the World Health

Assembly in May 2015 – offers the technical framework for all malaria-endemic countries.

This was designed to guide and support regional and national programs that aim to control

and eliminate malaria. The strategy establishes ambitious but achievable global objectives,

among which: reducing the incidence of malaria by at least 90% until 2030; reducing the

rates of mortality caused by malaria by at least 90% until 2030; eliminating malaria in at

least 35 countries until 2030; preventing the re-emergence of malaria in all countries that

are malaria-free.

This strategy was the result of an extensive consulting process carried out for 2 years and

which involved the participation of over 400 technical experts from 70 member states. The

strategy is based on three key pillars: to ensure universal access to the prevention, diagnosis

and treatment of malaria; to accelerate the efforts towards elimination and obtaining the

status of malaria-free country; to transform malaria surveillance into a basic intervention

(WHO - Global Technical Strategy for Malaria 2016-2030, 2015).

38

During the 1950s and 1960s, the campaigns for malaria eradication and control were a

success in countries with temperate climate or with reduced malaria cases. The emergence

of resistance to insecticides in mosquitoes and the resistance of the malaria parasite to

chloroquine have put an end to the period of malaria eradication, not to mention the weak

infrastructure in the areas of ongoing malaria as well as the decline in resources for research

on malaria (Lines, 1996; Carter and Mendis, 2002; Korenromp et al, 2003; ter Kuile et al,

2004). The international conference of 1992 re-established malaria control as a health

priority at global level. WHO declared in 1998 that poverty would be the main risk factor

concerning malaria (Lucas and McMichael, 2005). The funding planned for the 2-year

project (1997-1998) represented 12 times more than the contribution made by the WHO

during the previous decade. In 1998, the campaign Roll Back Malaria had launched

partnerships for basic techniques in malaria control strategies. In 2000 the United Nations

declared 2001-2010 to be the decade for Roll Back Malaria in the developing countries,

especially in Africa. In 2001 the resources for malaria control were amplified through the

creation of the Global Fund for fighting HIV, tuberculosis and malaria.

Malaria was eliminated in 5 countries from North Africa, namely Tunisia, Libya, Egypt,

Morocco, Algeria, where malaria was caused most frequently by P. vivax and transmitted by

mosquito species that were easier to control; but the risk of reintroducing malaria in these

countries is continuous. In the East, malaria is endemic in Madagascar, the Comoro Islands

(the Federal Islamic Republic of the Comoro Islands) and the French collectivity Mayotte and

Zanzibar. In Mauritius, malaria has been controlled well since 1950, but occasional malaria

outbreaks caused by the P. vivax species still occur, the last one after a cyclone in 1982.

Since then there has been a constant decrease of cases and the risk is now extremely low.

Since 1930 the Seychelles is free of malaria and it is thought that malaria vectors do not

exist there anymore (WHO/UNICEF - The Africa Malaria Report, 2003). A study carried out in

Africa, in West Kenya, showed that the main vector of malaria is Anopheles gambiae

(Fillinger et al, 2004) and the decrease in child mortality in this area is mainly achieved

through the use of insecticide-treated mosquito nets (Nevill et al, 1996).

In Asia, malaria represents a major problem in the Eastern Mediterranean sub-region,

especially in areas where in the last 30 years there have been emergency cases following

the deterioration of the health systems. Starting from 1998-1999, regional funds for malaria

control have increased. A study carried out in 2007 showed the effectiveness of neem oil (a

tree originating in West Asia) as a larvicide in malaria control (Okumu et al, 2007).

Considering resistance to synthetic insecticides built over the last years and their negative

effect on the environment, the use of neem oil may represent a cheap option for larvae

control. Used in various concentrations, it has proved to be a good larvicide, and the

average lethal concentration [LC (50)] of the composition against Anopheles stephensi, Culex

quinquefasciatus and Aedes aegypti was found to be 1.6, 1.8 and 1.7 ppm (Dua et al, 2009).

39

The main control strategies are access to quick and efficient treatment and the use of nets

treated with insecticide and biolarvicides (Mittal, 2003; WHO, 2006). Malaria with P.vivax is

endemic in Central Asia and Transcaucasia, and malaria with P. falciparum re-emerged in

Tajikistan in 1990, so starting from 2002 vector control strategies have been organized. A

degree of control of malaria epidemics has been achieved, although the report from 2003

showed an incidence of malaria 10 times higher than in 1990. South-East Asia is the region

with the highest resistance to medication in the world, contributing to the re-emergence of

malaria in many areas, especially along international borders. All the countries in South-East

Asia have adopted control strategies by using mosquito nets treated with insecticides and

larvicides. In Indonesia and Sri Lanka some very good results have been recorded, reducing

the incidence of malaria to its lowest level after 1967. In the West Pacific sub-region, where

malaria had re-emerged as a result of economic decline, large-scale population movement

and faulty sanitary systems, the control strategies implemented have yielded good results.

Malaria transmission has been reported in eight countries from Central America and the

Caribbean, as well as in the tropical forest area from the Amazon due to population

movement, mining and deforestation. All these areas have adopted control strategies based

on the use of mosquito nets treated with insecticides and larvicides, and therefore managed

to keep malaria epidemics under control. The use of mosquito nets treated with insecticides

came as a necessity after the resistance shown by Pl. falciparum to chloroquine (Pietra et al,

1991). Chloroquine has maintained its efficiency in the treatment and prophylaxis of malaria

with P. falciparum in Central America, north of the Panama Canal, the Dominican Republic

and Haiti, as well as in the treatment of malaria with P. vivax in the entire region. A new

strategy for creating new medication is the use of hybrid molecules that allow the

production of antipaludic molecules, such as trioxaquines, which are active against some

strains of infectious agents resistant to classic medication (Meunier and Robert, 2010).

Primaquine is the only medication that has an effect on the hepatic forms (hypnozoites), but

its use is controversial because it produces hemolysis in people with glucose-6-phosphate

dehydrogenase deficiency (Oliver et al, 2008; Bray et al, 2005). Primaquine may be used as

prophylaxis during travels and upon return from travels, as well as prophylaxis for persons

at a very high risk of being infected with P. vivax. Also, primaquine is administered to

persons suffering from P. vivax or P. ovale malaria in order to avoid the risk of a relapse,

more or less delayed because of the hypnozoites (Baird and Rieckmann, 2003; Baird et al,

2003; Baird and Hoffman, 2004; Ajdukiewicz, Ong, 2007; Carmona-Fonseca and Maestre,

2009).

For example, in Chimoio, Mozambique, there was a descending trend of malaria mortality

rates between 2010 and 2014, partially explained by the use of insecticide-treated mosquito

nets (Fig. 13) (Ferrão et al. 2017).

40

Fig.13. Malaria mortality trend in Chimoio, Mozambique, per month and year

(Source: Ferrão et al. 2017)

International funding for malaria control has increased in the last eight years, being

estimated at around 1.66 billion dollars in 2011 and 1.84 billion dollars in 2012. Funding

from the national government increased in 2011 to 625 million dollars. In spite of this, the

funds currently available for malaria prevention and control are noticeably below the level

of resources necessary to reach the global objectives regarding malaria. An estimated

amount of 5.1 billion dollars would be necessary annually between 2011 and 2020 so as to

allow universal access to interventions in case of malaria. In 2011, only 2.3 billion dollars

were available, less than half of what was necessary.

Malaria remains closely tied to poverty and the highest mortality rate is reported in the

poorest countries from Africa. Therefore, the global objectives for the control of malaria

cases and deaths will not be reached unless progress is made in the 17 countries targeted

where 80% of malaria cases are reported. Nigeria, the Democratic Republic of Congo, the

United Republic of Tanzania, Uganda, Mozambique and the Ivory Coast are the countries

with an estimated number of 103 million (47%) of malaria cases. In South-East Asia, the

second most affected region, India ranks first with 24 million malaria cases per year,

followed by Indonesia and Myanmar.

Malaria surveillance systems can detect arround 10% of the total number of malaria cases

worldwide in 41 countries in the entire world, but no reliable estimates can be provided

because of incomplete reports.

Malaria resistance to medication is of global interest in the fight to control P. falciparum

malaria (Girod et al, 2006). In four countries from South-East Asia resistance to artemisinin

was reported, and resistance to insecticides in 64 countries.

The funds allocated for malaria control had increased between 2004 and 2009 with very

good results, but they declined in the 2010-2011 period, a fact that could lead to reversing

41

the victories and giving malaria a new momentum instead. Thus, the number of mosquito

nets treated with insecticides in Sub-Saharan Africa decreased from 145 million in 2010 to

66 million in 2012. The malaria-control steps taken in the last eight years helped to avoid 1

million deaths. Nigeria and the Democratic Republic of Congo are the most affected

countries in Sub-Saharan Africa, while India is the most affected country in South-East Asia.

The 2012 World Malaria Report indicates that international funds for malaria seem to have

reached a level far below what is needed to reach the established objectives. An estimated

5.1 billion dollars are necessary every year between 2011 and 2020 in order to carry out all

interventions planned for the 99 countries with ongoing malaria transmission. While many

countries have increased internal funding for malaria control, the total available funds

worldwide remained at the level of 2.3 billion euro in 2011 – less than half of what is

necessary (WHO World Malaria Report, 2012).

Fig.14. Estimated number of deaths caused by malaria, 2002-2012

(Source: https://www.statista.com/chart/1758/estimated-number-of-deaths-caused-by-malaria-worldwide/)

42

PATHOGENESIS

Malaria infection develops in two phases: one that involves the liver (exoerythrocytic

phase), and one that involves red blood cells, or erythrocytes (erythrocytic phase). When an

infected mosquito pierces a person's skin to take a blood meal, sporozoites in the

mosquito's saliva enter the bloodstream and migrate to the liver where they infect

hepatocytes, multiplying asexually and asymptomatically for a period of 8–30 days (Bledsoe,

2005). After a potential dormant period in the liver, these organisms differentiate to yield

thousands of merozoites, which, upon rupture of their host cells, escape into the blood and

infect red blood cells to begin the erythrocytic stage of the life cycle (Bledsoe, 2005). The

parasite escapes from the liver undetected by wrapping itself in the cell membrane of the

infected host liver cell (Vaughan et al, 2008).

Within the red blood cells, the parasites multiply further, again asexually, periodically

breaking out of their host cells to invade fresh red blood cells. Several such amplification

cycles occur. Thus, classical descriptions of waves of fever arise from simultaneous waves of

merozoites escaping and infecting red blood cells (Bledsoe, 2005). Some P. vivax sporozoites

do not immediately develop into exoerythrocytic-phase merozoites, but instead produce

hypnozoites that remain dormant for periods ranging from several months (7–10 months is

typical) to several years. After a period of dormancy, they reactivate and produce

merozoites. Hypnozoites are responsible for long incubation and late relapses in P. vivax

infections (Richter et al, 2010) although their existence in P. ovale is uncertain (White,

2011).

The parasite is relatively protected from the attack by the body's immune system because

for most of its human life cycle it resides within the liver and blood cells and it is relatively

invisible to immune surveillance. However, circulating infected blood cells are destroyed in

the spleen. To avoid this, the P. falciparum parasite displays adhesive proteins on the

surface of the infected blood cells, causing the blood cells to stick to the walls of small blood

vessels, thereby sequestering the parasite from passage through the general circulation and

the spleen (Tilley et al, 2011). The blockage of the microvasculature causes symptoms such

as in placental malaria (Mens et al, 2012). Sequestered red blood cells can breach the blood-

brain barrier and cause cerebral malaria (Rénia et al, 2012).

43

CLINICAL MANIFESTATIONS Usually, patients with malaria become symptomatic a few weeks after the infection, but the

host’s previous exposure or host immunity against malaria could influence symptomatology

and the incubation period, which is typical for each Plasmodium species.

One important symptom is the typical recurrent fever (which may be periodic at 48 or 72

hours, depending on the species) (Fig. 15).

Fig.15. Malaria symptom - typical recurrent fever

(Source: https://www.dailystar.co.uk/health/592904/Malaria-symptoms-tablets-treatment-prevention

The classic paroxysm begins with a period of shivering and chills lasting for around 1-2 hours

and is followed by high fever. The excessive sweats experienced by the patient are followed

by the decrease of body temperature to normal or below normal. Virtually, all patients with

malaria experience headache. Other symptoms include tiredness, malaise, nausea and

vomiting, diarrhea, cough, shortness of breath, arthralgia and myalgia (Dondorp et al, 2017;

Nadjm et al, 2012; Bartoloni and Zammarchi, 2012) (Fig. 16, 17).

Fig.16. Malaria symptoms

(Source: https://www.lybrate.com/topic/symptoms-of-malaria-and-

chikunguniya/dd771f92ae9155433ef28c1ecc43adc7)

44

Many patients, particularly during the early infection, do not present the classic paroxysm

but may have several small fever spikes a day. Fever periodicity is specific for each species

(i.e., 48 h for P. falciparum, P. vivax, and P. ovale – so called tertian fever; 72 h for P.

malariae – also called quartan fever), but is not apparent during the initial infection

because of multiple broods emerging in the bloodstream, and is often not observed in P.

falciparum infections. Patients with long-standing, synchronous infections are more likely to

present classic fever patterns.

In severe cases, usually caused by P. falciparum, the following symptoms could be present:

jaundice, anemia, renal failure, shock, adult respiratory distress syndrome, confusion, coma,

encephalopathy, and acidosis (Dondorp et al, 2017; Nadjm et al, 2012; Bartoloni and

Zammarchi, 2012) (Fig. 17).

Fig.17. Malaria symptoms

(Source: http://zeenews.india.com/health/how-to-fight-dengue-malaria-flu-more-symptoms-prevention-

2046312

The World Health Organization (WHO) recommends that pregnant women should avoid

travelling to areas where there is a risk of malaria (WHO Information for travellers, 2018). If

women get malaria while pregnant, the mother and the baby are at increased risk of

developing serious complications such as: premature birth (birth before 37 weeks of

pregnancy), low birth weight, restricted growth of the baby in the womb, stillbirth,

miscarriage, and even mother death.

45

DIAGNOSTIC Malaria must be promptly recognized in order to treat the patient effectively and in time,

and in order to control the spreading of the infection in the community through the local

mosquitoes. Delaying the diagnosis and the beginning of the treatment represents the main

cause of death in patients with malaria.

Malaria may be suspected on the basis of the patient’s history of recent travel or living in

malaria-endemic areas, of their symptoms and of the findings from clinical examination.

However, in order to confirme the diagnosis, there should be done laboratory tests that

would demonstrate the presence of the malaria parasites.

There could be a problem in countries were malaria is no longer endemic because doctors

may not be familiar with the disease. Clinicians who see a patient with malaria may not take

into account malaria as one of the possible diagnoses and therefore not recommend the

necessary diagnostic tests. The laboratory may not have the required experience with

malaria and may not be able to detect the parasites during examination of blood smears

under the microscope.

CLINICAL DIAGNOSTIC The patient’s history should include inquiries into the following: recent or remote travel to

an endemic area; immune status, age, and pregnancy status; allergies or other medical

conditions; medications currently being taken.

Most of the patients with malaria have no specific physical findings, but splenomegaly may

be present. Symptoms of malarial infection are nonspecific and may manifest as a flu-like

illness with fever, headache, malaise, fatigue, and muscle aches. Some patients with malaria

present diarrhoea and other gastrointestinal symptoms. Immune individuals may be

completely asymptomatic or may present mild anemia. Nonimmune patients may quickly

become very ill. In general, however, the occurrence of fever periodicity is not a reliable clue

for the diagnosis of malaria.

Severe malaria primarily involves the P. falciparum infection, although death due to splenic

rupture has been reported in patients with non-P. falciparum malaria. Severe malaria

includes cerebral malaria (sometimes with coma); severe anemia; respiratory abnormalities

(with metabolic acidosis, associated respiratory distress, and pulmonary edema); signs of

malarial hyperpneic syndrome (alar flaring, chest retraction, use of accessory muscles for

respiration, and abnormally deep breathing); renal failure (usually reversible) (Dondorp et

al, 2017; Bartoloni and Zammarchi, 2012).

46

In children, malaria has a shorter evolution, often rapidly progressing to severe malaria.

Children are more likely to present hypoglycemia, seizures, severe anemia, and sudden

death, but they are much less likely to develop renal failure, pulmonary edema, or jaundice

(Bartoloni and Zammarchi, 2012).

In some malaria-endemic areas, transmission is so intense that a large part of the

population is infected, but does not become ill. These types of carriers develop sufficient

immunity to protect them from malaria.

Malaria is clinically suspected on the basis of fever or a history of fever, but antimalarial

treatment should be limited to cases with positive tests. Patients with negative results

should be reassessed for other common causes of fever and treated appropriately. In

patients with suspected severe malaria and in other high-risk groups, such as patients living

with HIV/AIDS, absence or delay of parasitological diagnosis should not delay the immediate

start of antimalarial treatment.

LABORATORY DIAGNOSTIC The following blood investigations should be performed:

blood culture, hemoglobin concentration, platelet count, investigation of liver and

renal function, electrolyte concentrations (especially sodium), monitoring of

parameters suggestive of hemolysis (haptoglobin, lactic dehydrogenase,

reticulocyte count);

in selected cases, rapid HIV testing is recommended;

white blood cell count: leukocytosis is present in less than 5% of malaria patients

(in this case the differential diagnosis should be broadened);

G6PD level has to be checked if the patient is to be treated with primaquine;

the glucose level has to be evaluated to rule out hypoglycemia if the patient has

cerebral malaria.

The following imaging investigations may be considered:

chest radiography if respiratory symptoms are present;

Computed Tomography of the head if central nervous system symptoms are

present.

Specific tests for malaria infection should be carried out:

microhematocrit centrifugation (sensitive but incapable of speciation);

fluorescent dyes/ultraviolet indicator tests (may not yield speciation information);

the standard tests: thin (qualitative) or thick (quantitative) blood smears (1

negative smear does not exclude malaria as a diagnosis and several more smears

should be examined over a 36-hour period);

47

alternatives to blood smear testing (used if the blood smear expertise is

insufficient) are rapid diagnostic tests, polymerase chain reaction assay, nucleic

acid sequence-based amplification, and quantitative buffy coat (Bartoloni and

Zammarchi, 2012; Greenwood et al, 2005).

All malaria suspect patients should be treated on the basis of a confirmed diagnosis through

microscopic examination or by a rapid diagnostic test (RDT) (immunochromatography) that

aim to detect the antigens in the blood (Fig. 18 ). However, before using these rapid tests on

a large scale, their accuracy should be improved. It is recommended that all RDTs should be

followed by microscopy in order to confirm the diagnosis and, if positive, in order to

quantify the proportion of the red cells from the infected blood. These rapid tests may

reduce the necessary time for malaria diagnosis.

Fig.18. Rapid Diagnostic Test for Malaria

(Source: http://adulldayatwork.blogspot.com/2011/10/25-october-2011-tuesday-rapid-malaria.html

Molecular identification of the malaria parasite represents the certain diagnostic method,

but the Polymerase Chain Reaction (PCR) results are not always available fast enough in

order to be of great value in establishing the diagnosis of infection with Plasmodium (Fig.

19). The PCR is most useful for the confirmation of the parasite species after the diagnosis

was done either by microscopy of thick drops or by RDT.

48

Fig.19. PCR test for malaria identification

(Source: Ivănescu, 2015)

Serology detects antibodies against malaria, using either the indirect immunofluorescence

(IFA) or the ELISA test. Serology does not detect the current infection, but measures all past

exposure. Malaria parasites can be detected using microscopic hematological examination

(thick peripheral blood drops) (Fig. 20).

Fig.20. Thick peripheral blood drops examination

(Source: https://labmedicineblog.com/2018/06/19/hematology-case-study-a-51-year-old-woman-with-fever-

and-chills/

Before the examination, the specimen is often coloured with Giemsa coloration (Fig. 21).

This technique remains the golden standard for laboratory malaria confirmation. However,

it depends on the quality of the reagents, the microscope, and the laboratory’s experience.

49

Fig.21. Hematologic diagnosis of malaria

(Source: Personal collection)

The malaria suspected cases from Africa are increasingly verified by using a rapid test before

prescribing antimalarial treatment, thus ensuring an efficient use of resources available for

the elimination of the parasites, although the main cause of patient fever remains unknown

because of the potential of a co-infection with other febrile illness. The large-scale use of

the rapid tests does not necessarily preclude the over evaluation of clinical malaria cases or

the management of the sub-optimal cases of febrile patients. In a recent publication from

the journal eLife, a new approach was presented that allows the deduction of the spatio-

temporal prevalence of the malarial fever associated with Plasmodium falciparum, as well

as of the non-malarial fever presented by children from Sub-Saharan Africa between 2006

and 2014. It is estimated that 35.7% of the febrile cases reported by the author were

accompanied by malaria infection in 2014, but only 28.0% of these (10.0% of all febrile

cases) were caused by malaria. The majority of the febrile cases among children with

positive malaria are therefore caused by non-malarial diseases (Fig. 22).

Also, anemia or the decrease of blood hemoglobin concentration levels is frequently

associated with malaria. Anemia is very prevalent in Sub-Saharan Africa, especially among

children and women at the reproductive age, but so far, it has not been quantified in order

to establish the contribution of malaria, nor the contribution of other causes to the onset of

anemia (for example, the iron deficiency diet, helminth infection) (Dalrymple et al, 2017).

50

In collaboration with the ROAD-MAP team, data have been collected from the survey done

in households where respondents are asked about their fever and treatment history and

receive a diagnosis based on malaria parasites and the measurement of their hemoglobin

concentration in the blood. Also, the data from the survey are associated with the distance-

sensitive variables collected by ROAD-MAP in order to help with the spatial prediction of the

measurements of interest.

Fig.22. The proportion of malaria positive fever from all causes of fever

with children under 5

(Source: https://elifesciences.org/articles/29198)

It has therefore been noted that prior to using the rapid tests on a large scale, antimalarial

treatment used to be administered in high proportions, and an inaccurate diagnosis of this

disease was documented.

Moreover, not all the people that develop malaria request treatment in public health

institutions. Some of them receive treatment in units that do not report malaria cases, such

as private medical institutions, while others do not get treated. This contributes to under-

reported malaria cases. Before the administration of any treatment it is recommended that

patients have done all the tests for evaluating resistance to medication. These tests take

place in specialized laboratories which assess the susceptibility of the parasites found in a

certain patient to antimalarial compounds. There are two main laboratory methods:

in vitro tests: the parasites are grown in culture in the presence of an

increasing concentration of medication; the concentration of the medication

that inhibits the growth of the parasite is used as a final step;

molecular characterization: the molecular markers evaluated through PCR or

genetic sequencing also allow to predict, to a certain degree, resistance to

some medication.

51

PROPHYLAXIS In 2016 the governments of the malaria-endemic countries and their international partners

invested over 2.7 billion dollars in malaria control measures and eradication. The African

region takes up 74% of the budget, the percentages decreasing for the regions in South-East

Asia (7%), Eastern Mediterranean and America (each 6%) and West Pacific (4%). The

governments of the endemic countries contributed only 800 million USD, which represents

just 31% of the invested sum, a fact that shows that in the absence of involvement of

partners from non-endemic countries, the fight to eradicate malaria would have been lost

from the start. The United States of America (USA) has offered one billion dollars (38%),

followed by an important contribution from the United Kingdom of Great Britain and

Northern Ireland (Great Britain), as well as France, Germany and Japan (WHO - World

Malaria Report, 2017).

At the 2010 annual meeting, "Global Technical Strategy for Malaria 2016–2030", an annual

budget was established until the year 2020 in order to decrease the incidence of malaria

cases and the rate of mortality by at least 40%, a budget that reaches 6.5 billion dollars per

year. The deficit causes concern since it is not even sufficient for maintaining the level

reached until now.

First line prevention consists in protection against mosquito bites of the Anopheles genus.

Prophylactic measures involve sleeping under nets treated with long-lasting insecticide and

the use of protective clothing and insect repellents (Fig. 23).

In 2016, the percentage of households that used mosquito nets treated with insecticide had

increased up to 80% compared to the 50% level registered in 2010; however, there is a

periodic decline when countries change insecticides or take them by rotation, using more

expensive ones. The residual spraying of indoor spaces with insecticide is a strong means of

rapidly reducing malaria transmission. Its potential is achieved when at least 80% of the

houses from the targeted areas are sprayed. Indoor spraying is efficient for 3-6 months,

depending on the insecticide and the type of surface sprayed. In some situations, more

rounds of spraying are needed in order to protect the population for the entire malaria

season.

52

Fig.23. Long-lasting insecticide-treated mosquito nets

(Source: http://www.who.int/immunization/programmes_systems/interventions/malaria_llins/en/)

Depending on the risk of malaria transmission in the area to be visited and the species of

Plasmodium that exist, travellers may also take prophylactic medication (chemoprophylaxis)

before, during and upon returning from the journey.

Some categories of travellers, especially small children, pregnant women and persons with a

weak immune system, present an increased risk of developing serious forms of the disease,

if they were to contract it. In pregnant women, malaria increases the risk of maternal death,

spontaneous miscarriage, the birth of a dead baby or a baby with a low weight at birth, as

well as the associated risk of neonatal death. Because of this, pregnant women should avoid

travelling to malaria-endemic countries, and parents are advised to not take infants or

yuong children in areas where there is the risk of infection with the P. falciparum strain.

When travel cannot be avoided it is important that travellers take effective preventive

measures against malaria, even when they travel to areas with transmission of malaria

caused by P. vivax. In 2012, WHO recommended seasonal chemoprevention of malaria as an

additional measure of preventing malaria for the areas from the African sub-region of Sahel.

The strategy involves monthly administration of amodiaquine and sulfadoxine

pyrimethamine to all children under the age of 5 during the high-transmission season. It is

recommended that travellers consult all national centers for fighting against the disease or

other institutions that can offer information regarding preventive measures that must be

taken before going to malaria-endemic countries or regions.

Prevention of drug resistance or delaying its occurrence is essential for the success of both

strategies – national and global – for malaria control and eradication. In order to contribute

to the protection of current and future antimalarial medication, all malaria episodes should

53

be treated with combined therapy and antimalarial medication shoud be administered in

optimal doses. All cases of suspected malaria should be confirmed through RDT or

parasitological tests.

VECTOR CONTROL A big part of the success in fighting malaria is due to vector control. Vector control largely

depends on the use of pyrethroids, which are the only class of insecticides recommended at

present.

In the last years, resistance of mosquitoes to pyrethroids has grown in many countries. In

some areas, a resistance to all 4 classes of insecticides used for public health has been

detected. Fortunately, this resistance was only rarely associated with decreased efficiency of

mosquito nets treated with insecticides with prolongued action, which continue to offer a

substantial level of protection in the majority of cases. The use, in turn, of different classes

of insecticides for indoor spraying is recommended as an approach for the management of

resistance to insecticides.

However, the malaria-endemic areas from Sub-Saharan Africa and India raise significant

concerns because of high levels of malaria transmission and recent reports regarding the

spread of areas with resistance to insecticides. Using two different insecticides on a

mosquito net is an opportunity to reduce the risk of development and spread of resistance

to insecticides; the further development of these new nets is a priority.

Detection of resistance to insecticides should be an essential component of all national

efforts for malaria control in order to ensure that the most efficient methods of vector

control are used. Choosing the insecticide for indoor residual spraying (IRS) should always

depend on recent and local data about the susceptibility of target vectors.

The mosquitoes of the Anopheles genus are found all over the world with the exception of

the arctic area, while the anopheles species is found not only in malaria-endemic regions,

but also in areas where malaria had been eradicated. In order to avoid the risk of spread of

a number of vector-borne diseases, the transport of vectors must be controlled; therefore,

the transfer of mosquitoes as a result of trade on tyres or in containers must be prevented.

Also, infected people (tourists and immigrants) with pathogenic agents transmitted through

vectors must be diagnosed and treated as quickly as possible, since the diseases transmitted

by mosquitoes have no borders, and their spread is also a consequence of people mobility

and globalization. Therefore, increased mobility of people and international commerce play

a more important role in the spread of vectors and their pathogenic agents / parasites than

the increase in temperature (Yi et al, 2014).

54

The data collected from 76 malaria-endemic countries for the period 2010-2016 showed

that resistance to at least one insecticide was reported in 61 countries. Resistance to two or

more classes of insecticides was reported in 50 countries. Resistance to pyrethroides – the

only class of insecticides currently used in treating the mosquito nets, is largely spread in all

areas, and the percentage of countries that reported resistance to insecticides in 2016

increased to 81%, as compared to 71% in 2010 (WHO - World Malaria Report, 2017).

Despite the resistance evidenced, mosquito bed nets treated with insecticide remain the

most efficient instrument used in malaria prevention, even in areas where resistance to

pyrethroides has been reported.

PREVENTION OF MALARIA IN HUMANS At this time prevention and control of malaria includes three directions:

reducing the contact between the vector and the human host,

special prevention with antimalarials, and

early diagnosis and appropriate treatment of malaria episodes to limit

transmission (Heymann, 2012; Dondorp, 2017).

GENERAL PREVENTION - MINIMIZING THE CONTACT BETWEEN THE HUMAN HOST AND

THE MOSQUITO

A very important method of general prevention for malaria in endemic areas is the

use of mosquito bed nets treated with insecticide (ITN). They have to be retreated

after 3 washes or at least once a year. An improved variant are long-lasting

insecticide-treated mosquito nets (LLINs) incorporating pyrethroids (Heymann,

2012; Ouattara et al, 2014). There are new hopes regarding the use of mosquito

nets with two insecticides to reduce the risk of resistance (Heymann, 2012).

Another general prevention method is indoor residual insecticide spraying (IRS),

which targets the adult mosquito population and has a high rate of success when

more than 80% of the houses in the area are sprayed (WHO - Malaria, 2018;

Heymann, 2012). This method, which is a public health intervention, has

maximum efficacy when mosquitoes remain indoors on surfaces that can be

sprayed, when people are exposed in the house or in the vicinity of the house,

when the method is applied before the start of the transmission season or before

the peak transmission period (Heymann, 2012; Salam et al, 2014). At the moment,

there are 12 WHO recommended insecticides for this measure, and the choice of

these substances is dependent on the susceptibility of the insecticide, the

peculiarities of the vector, the degree of safety for humans and the environment,

the efficacy and the cost-effectiveness ratio. The DDT is still used to control the

Anopheles species, in line with the Stockholm Convention on Persistent Organic

Pollutants which prohibits the use of DDT, except for the purpose of public health

insurance (Heymann, 2012).

55

Controlling larval stages by eliminating mosquito reproduction sites has limited

value in most areas where malaria transmission persists. Such a measure is

represented by filling and draining or increasing the flow of water in natural or

artificial channels. Chemical or biological control methods, such as bacterial

larvicides or larvivorus fish, can be difficult to implement in rural areas, but some

African urban areas have been successful. These methods are useful adjuvants in

arid, coastal and urban areas, but methods for maintaining a low level of

responsiveness may be necessary in areas where malaria elimination has been

achieved (Heymann, 2012; Bocsan et al, 1999).

Intermittent preventive use of a full dose of antimalarial at predetermined

intervals in trimesters 2 and 3 of pregnancy is an effective measure for reducing

malaria in pregnant women in areas with a P. falciparum stable transmission, with

moderate to intensive intensity such as Africa. The method has limited use in

other parts of the world, where transmission is unstable and of low intensity.

Also, in endemic areas, using ITN and LLIN for this category of people diminishes

the effects of malaria during pregnancy (Heymann, 2012; Mpogoro et al, 2014).

Another general prevention measure is the health education of people travelling

to endemic areas so that they are aware what regions may pose a risk, what are

the protective measures to be taken and how to manage feverish syndromes if

they occur after coming back (Luca, 2002).

SPECIAL PREVENTION USING ANTIMALARIAL DRUGS

Before travel chemoprophylaxis should include recommendations for the use of the

appropriate antimalarial medication (if any) for the chosen destination, as follows

(Heymann, 2012; Bocsan, 1999; Luca, 2002):

In areas with a very low risk of malaria transmission, chemoprophylaxis may not be

recommended, taking into account that the risk of side effects associated with

antimalarials may outweigh the potential benefits. But the travellers should be aware

of the possibility of malaria if they develop a febrile illness.

Chloroquine chemoprophylaxis can be used in areas where only P. vivax malaria

occurs and those areas where P. falciparum is susceptible to chloroquine. 5 mg base /

kg / week is recommended at single dose or 10 mg base / kg / week, divided into 6

doses / day (Heymann, 2012; Luca, 2002).

In areas where there is a risk of transmission for P. vivax and P. falciparum and there

is resistance to chloroquine, chloroquine chemoprophylaxis should be combined with

proguanil (3 mg / kg / day).

In areas of high risk for P. falciparum malaria and resistance to antimalarial drugs,

elective chemoprophylaxis is performed with atovaquone-proguanil (250 mg

atovaquone and 100 mg proguanil / day), doxycycline (1.5 mg / kg / day) or

mefloquine (5 mg / kg / week). These recommendations also apply to areas with

56

moderate or low risk of P. falciparum malaria, but with high levels of resistance to

antimalarial medication (Heymann, 2012; Luca, 2002).

The use of daily antimalarials (atovaquone-proguanil, chloroquine, doxycycline,

proguanil) should be started one day before arrival in the risk area. With regard to

chloroquine, administration should be started one week before arrival and for

mefloquine with 2-3 weeks before departure to achieve high, effective antibody

levels and to detect possible side effects before departure.

Preventive drug administration should be strictly used throughout the journey in

areas at risk of malaria and should be continued for 4 weeks after the last possible

exposure (Heymann, 2012; Luca, 2002).

SPECIFIC PREVENTION - VACCINE DEVELOPMENT

Although there are promises of a vaccine against malaria, there is currently no licensed

population-based vaccine. he most advanced is the research for the development of a

candidate vaccine against P. falciparum, a preparation known as RTS, S/AS01 (a

recombinant vaccine using pre-erythrocyte stage proteins) - Mosquirix. This vaccine was

evaluated in an extended clinical trial conducted in 7 African countries and was positively

evaluated by the European Medicines Agency in July 2015 (WHO - Malaria, 2018; Dondorp,

2017; Umeh et al, 2014). The vaccine efficacy against malaria with clinical manifestations in

children aged 5 to 17 months was 53% (CI95% = 28-69%), evaluated over a period of 8

months. For the older child the efficacy of this vaccine was lower. Other candidate vaccines

include the attenuated whole-parasitic vaccine, which provides a high level of protection,

but raises problems of production and administration. Other types of candidate vaccines

target parasite stages which can be found in the bloodstream or asexual stages of

Plasmodium (Dondorp, 2017). WHO strongly supports these pilot studies as an important

step in obtaining the world's first antimalarial vaccine, so in October 2015 it recommended

the implementation of the pilot studies for RTS, S/AS01 vaccine in a limited number of

African countries. In November 2016, WHO announced that the safety and efficacy of the

RTS, S/AS01 vaccine would be evaluated in two pilot studies (Phase III clinical trials)

conducted in 3 Saharan African countries (Ghana, Kenya and Malawi) that were scheduled

to start at the begining of 2018 (WHO - Malaria, 2018). This pilot project could open the way

for a larger development of the vaccine if its safety and efficacy are considered acceptable.

THE PREVENTION OF P.VIVAX OR P.OVALE INFECTION RELAPSES In order to prevent the relapses, primaquine for 14 days (0.25-0.5mg / kg body weight) in all

transmission environments is the recommendation for malaria caused by P.vivax or P.ovale

in children and adults (with the exception of pregnant women and infants before the age of

6 months). In patients with G6PD deficit, the prevention of relapses is made with

primaquine in a dose of 0.75mg / kg body weight once a week, for 8 weeks, under strict

57

medical surveillance because of the hemolysing potential induced by primaquine (WHO -

Guidelines for the Treatment of Malaria, 2015).

Artemisinin and its derivatives must not be used as oral monotherapy because they could

produce artemisinin resistance. The WHO recommends regular monitorization of

antimalarial medication efficiency in order to ensure that the selected treatments remain

efficient. These actions have to be implemented by the national programs for malaria

control. Resistance to antimalarial medication represents a recurring problem. The

resistance of P. falciparum to previous drugs generations, such as chloroquine and

sulfadoxine pyrimethamine (SP), had become largely spread in the 1950s and 1960s,

undermining malaria control efforts and decreasing the chances of survival in children. The

WHO recommends routine monitoring of resistance to antimalarial medication and supports

countries in consolidating their efforts in this regard. In the past years, resistance of

parasites to artemisinin was detected in 5 countries from the Mekong subregion: Cambodia,

Lao People's Democratic Republic, Myanmar, Thailand and Vietnam. Studies have confirmed

that resistance to artemisinin has appeared independently in many areas of these

subregions.

In 2013, the WHO launched an emergency answer to resistance to artemisinin (ERAR) from

the Mekong subregion, a high-level action plan in order to limit the spread of parasites that

are resistant to medication and offer instruments that can help the population exposed to

the risk of malaria infection. In parallel, there were reported some cases of high resistance

levels to medication that was associated with ACT. A new approach was necessary in order

to keep up with the changes in the malaria landscape. As a consequence, the Advisory

Committee for policies in the domain of malaria of the WHO recommended in September

2014 the adoption of the objective to eliminate the malaria caused by P. falciparum in this

subregion until 2030. The WHO launched the Strategy for the elimination of malaria in the

Mekong subregion (2015-2030) in 2015, which was approved by all the countries from the

subregion. With technical guidance from the WHO, all GMS countries have elaborated

national plans for the elimination of malaria. Together with partners, the WHO offers

permanent support to all countries in their efforts to eliminate malaria through the

dedicated program of elimination of malaria in Mekong, a new initiative that evolved from

ERAR (WHO - Strategy for malaria elimination in the Greater Mekong Subregion: 2015-2030,

2015).

58

TREATMENT

Malaria is an entirely preventable and treatable disease. The primary objective of the

treatment is to ensure the rapid and complete elimination of the Plasmodium parasite from

the patient’s blood in order to prevent the progression from uncomplicated malaria to

severe disease and to prevent the chronic infection that leads to severe anemia. From a

public health perspective, the goal of the treatment is to reduce transmission of the

infection to other people by reducing the infectious reservoir, and to prevent the spread of

the antimalarial drug resistance.

In patients with suspected malaria, the parasitological diagnosis must be confirmed before

actually beginning the treatment for malaria, either by microscopy or by a rapid diagnostic

test. The treatment based only on clinical manifestations has to be used solely for the cases

in which the diagnostic tests are not accessible upon 2 hours from the arrival of patients for

treatment. In order to prevent any life threatening complications the treatment with an

efficient and sure antimalarial drug has to be used within 24 hours from the onset of the

fever.

Malaria is treated with antimalarial medications, but the selected drugs depend on the type

and severity of the disease. Also, medications against fever are commonly used in malaria,

but their effects on outcomes are not very clear (Meremikwu et al, 2012; Amadi et al, 2015).

Simple or uncomplicated malaria may be treated with oral medications. Artemisinins in

combination with other additional antimalarials (including: amodiaquine, lumefantrine,

mefloquine or sulfadoxine/pyrimethamine), also known as the artemisinin-combination

therapy (ACT), is the most effective treatment for the P. falciparum infection that decreases

the resistance to any single drug component (WHO - Guidelines for the Treatment of

Malaria, 2015; Amadi et al, 2015; Kokwaro, 2009). Another recommended combination is

dihydroartemisinin and piperaquine. ACT is about 90% effective when used to treat

uncomplicated malaria (WHO - Guidelines for the Treatment of Malaria, 2015; Amadi et al,

2015; Howitt et al, 2012). To treat uncomplicated malaria during pregnancy, WHO

recommends the use of quinine plus clindamycin early in the pregnancy (1st trimester), and

ACT in later stages (2nd and 3rd trimesters) (WHO - Guidelines for the Treatment of Malaria,

2015; Piola et al, 2010; McGready et al, 2012). Infections with P. vivax, P. ovale or P.

malariae usually do not require hospitalization. Treatment of P. vivax should include both

treatment of blood stages (with chloroquine or ACT) and clearance of liver forms with

primaquine (WHO - Guidelines for the Treatment of Malaria, 2015; Meremikwu et al, 2012).

Treatment with tafenoquine prevents relapses after confirmed P. vivax malaria (Kokwaro,

2009; Rajapakse et al, 2015).

In areas with low transmission, one single small dose of primaquine should be used in the

antimalarial treatment with the purpose of reducing transmission of the infection. The G6PD

59

testing is not necessary because one single small dose of primaquine is efficient in blocking

transmission and it is less likely to cause severe toxicity for subjects with G6PD deficit.

The infection with P. falciparum almost always causes severe and complicated malaria, but

the other species usually cause only febrile disease (White, 2011). Severe and complicated

malaria are medical emergencies that produce high mortality rates (between 10% and 50%)

(Howitt et al, 2012). Cerebral malaria is the form of severe and complicated malaria with the

worst neurological symptoms (Manyando et al, 2011). Recommended treatment for severe

malaria is the intravenous use of antimalarial drugs. For severe malaria, parenteral

artesunate is superior to quinine in both children and adults (WHO - Guidelines for the

Treatment of Malaria, 2015; Sinclair et al, 2012). Other studies show that artemisinin

derivatives (artemether and arteether) have been as efficacious as quinine in the treatment

of cerebral malaria in children (Rajapakse et al, 2015).

It is essential that full doses of effective parenteral (or rectal for children under the age of 6

years) antimalarial treatment be given promptly in the initial treatment of severe malaria (at

least for 24 hours). This should be followed by a full dose (3 days administration) of effective

oral ACT if the patient can tolerate the drug. Two classes of medicines are available for

parenteral treatment of severe malaria: artemisinin derivatives (artesunate or artemether)

and the Cinchona alkaloids (quinine and quinidine). Parenteral artesunate is the treatment

of choice for all severe malaria. A large randomized clinical trial regarding the treatment of

severe falciparum malaria showed an important reduction in the mortality rate with

parenteral (intravenous or intramuscular) artesunate as compared with parenteral quinine.

The artesunate was simpler and safer to use, with no increase in neurological sequelae in

artesunate-treated survivors (WHO - Guidelines for the Treatment of Malaria, 2015).

The most used drugs in malaria treatment are active against the parasite forms in the blood

(the form responsible for the occurrence of the disease) and include: chloroquine;

atovaquone-proguanil (Malarone®); artemether-lumefantrine (Coartem®); mefloquine

(Lariam®); quinine; quinidine; doxycycline (used in combination with quinine); clindamycin

(used in combination with quinine), and artesunate (Bartoloni and Zammarchi, 2011;

Greenwood et al, 2005).

60

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SOURCES FOR FIGURES AND TABLES - ADDITIONAL REFERENCES: 1. http://www.microbiologynotes.com/life-cycle-of-plasmodium-vivax/

2. https://ecdc.europa.eu/en/disease-vectors/facts/mosquito-

factsheets/anopheles-sacharovi

3. http://mosquito-taxonomic-inventory.info/file/5212

4. https://cameronwebb.wordpress.com/tag/anopheles-gambiae/

5. http://wwwnc.cdc.gov/travel/yellowbook/2012/chapter-3-infectious-

diseases-related-to-travel/malaria.htm

6. https://www.statista.com/chart/1758/estimated-number-of-deaths-

caused-by-malaria-worldwide/

7. https://www.dailystar.co.uk/health/592904/Malaria-symptoms-tablets-

treatment-prevention

8. https://www.lybrate.com/topic/symptoms-of-malaria-and-

chikunguniya/dd771f92ae9155433ef28c1ecc43adc7

9. http://zeenews.india.com/health/how-to-fight-dengue-malaria-flu-

more-symptoms-prevention-2046312.

10. http://adulldayatwork.blogspot.com/2011/10/25-october-2011-

tuesday-rapid-malaria.html

11. https://labmedicineblog.com/2018/06/19/hematology-case-study-a-51-

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12. https://elifesciences.org/articles/29198

13. http://www.who.int/immunization/programmes_systems/interventions

/malaria_llins/en/