Parasitol Res (2009) 105:609627 DOI 10.

1007/s00436-009-1524-8

REVIEW

Seventy-five years of Resochin in the fight against malaria

Markus Jensen & Heinz Mehlhorn

Received: 1 June 2009 / Accepted: 8 June 2009 / Published online: 11 July 2009 # Springer-Verlag 2009

Abstract The four different forms of human malaria have threatened humanity since time immemorial and to this day, they exact a death toll of one to three million people annually. Synthetic anti-malarial agents have been in development since early 1900. Perhaps the most successful and widely used drug, Resochin (chloroquine), was discovered 75 years ago; for a long time, it was the drug of choice and to this day, it is still used in many regions of the world as a reliable treatment against simpler forms of malaria. In regions where it has not been in use against malaria tropica for quite some time due to the development of resistances, it has regained some of its efficacy. This review traces the discovery and the mechanism of action of this substance, illustrates the significance of malaria today, and underlines the need for controlled and reliable therapeutic measures.

Malariaa worldwide epidemic History and significance Plagues threaten humans since their early daysnowadays with increasing importance (Hacker 2003; Grntzig and Mehlhorn 2005). Malaria, which comes from the Italian/ Latin (mala aria = bad air), is still the most significant
M. Jensen Bayer Vital GmbH, Kaiser Wilhelm Allee, 51368 Leverkusen, Germany e-mail: Markus.Jensen@bayerhealthcare.com H. Mehlhorn (*) Institut fr Parasitologie der, Heinrich-Heine-Universitt, 40225 Dsseldorf, Germany e-mail: mehlhorn@uni-duesseldorf.de

tropical disease to this day. Approximately one million children (especially children <5 years of age) die of this epidemic each year out of an annual total of some 247 million deaths worldwide (WHO 2008). That translates to at least 2,0003,000 victims every day, whose fate is barely acknowledged in the press, while even the tiniest local misfortune receives widespread public attention through the media. Even though malaria only affects humans and thus there seemed to be a good chance of eradicating it, its status as a major killer has remained largely unchanged. This is due to various factors. While the WHO has made tremendous efforts to eradicate malaria since the Second World War, spending many billions of euros, this disease continues to exist in 109 countries (WHO 2008). This is mainly due to the fact that the vector mosquitoes in these mostly tropical and economically underdeveloped countries are active throughout the year, while at the same time, any attempts at proactively and comprehensively controlling the epidemic have proven to be a disaster. As a result, lots of mosquitoes combine with lots of infected people present a situation in these countries, in which the mosquitoes end up of passing along the single-cell pathogen to other mosquitoes. The links between the transmission of malaria and the symptoms of the disease were first discovered in the period of 18801898, especially through the research of Alphonse Laveran (18451922), Ronald Ross (18571932), Patrick Manson (18441922), and Giovanni Battista Grassi (1854 1925), although the disease has likely accompanied the evolution of humanity since the Middle Pleistocene (780,000120,000 years ago; Gaham 1966; Mattingly 1976). There are specific reports concerning tertian fever from the ancient Assyrians and Babylonians (circa 2700 B.C. and 2000 B.C., respectively). Even the Greek physician Hippocrates (460377 B.C.) was already making a distinction between the 3- and 4-day fever. An early connection

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was made between this fever and swamps. The Greek philosopher Empedocles (483423 B.C.) was successful in combating malaria when he had two small rivers near the village of Selinunte redirected through a swampy area, thus reducing the breeding grounds for mosquitoes. This knowledge was passed on to the Romans, such as Plinius (2379 A.D.), who was skeptical of ever finding any type of medicinal treatment for the disease, but who considered pepper to be effective. Malaria, which does not discriminate based on social standing, has carried off not only many nameless millions but also many famous people as well, such as Alexander the Great, German emperors, and chancellors (for example, the Archbishop of Cologne, Rainald von Dassel [11201167]during the siege of Rome by Kaiser Frederick I), famous military commanders, and scientists, thus changing the course of human history at many points in time. As a result, malaria is by all means a social epidemic, which does not spare even the rich and powerful. In order to give an idea of the dimension of the economic impact of malaria, the WHO pointed out in the introduction to World Health Day 2003: Africas gross domestic product today would be roughly 100 billion dollars higher if malaria had been successfully eradicated 30 years ago (WHO 2003). Geographic prevalence The prevalence of malaria, which is caused by four different single-cell parasites in humans (see below), is linked to certain factors, which are only to be found in a few regions of the world: water and heat. Water (of a certain degree of purity) is necessary because it forms the habitat for the development of the vector mosquitoes. Freshwater is the preferred habitat, but several species of mosquito have adapted to brackish water (spray zone water). In addition, constant heat is also necessary because the parasites will not reach maturity and become infectious within the cold-blooded vector mosquitoes unless the outside temperature remains above 1618C for several days. Based on these reasons, malaria is mostly limited nowadays to tropical and subtropical zones. This was not always the case. After the Second World War, malaria also occurred within swampy areas or in humid lowlands in relatively temperate zones (for example, in northern Italy or even in southwestern Germany and North RhineWestphalia, along the Ruhr river). However, the pathogenic foci within these regions have now disappeared because any infected persons received rapid medical treatment and the swamp areas themselves have been drained; while suitable mosquito species are still present, their numbers are low. As a result, people within temperate zones are only at risk for infection if they travel to affected regions; however, these regions are often dream destinations for many

tourists, due to the beauty of their natural surroundings. These tourists are then especially at risk due to a lack of semi-immunity, which the local populations have acquired, as well as a lack of awareness of the problem (it is in the economic interest of the tourism industry to make light of the risk). Usually, however, people do not fall ill or experience serious symptoms until they have returned to their home countries. Due to a lack of experience on the part of the local health care professionals in dealing with this human parasitosis, misdiagnosis-related complications may occur; these can have life-threatening consequences often resulting in death, especially in the case of malaria tropica parasites (Plasmodium falciparum, see below), as evidenced by the ten to 15 fatalities each year in Germany. Vectors of malaria parasites The females of approximately 70 out of 400 species of the genus Anopheles (Greek anophelos = harmful, useless), 58 mm in size, serve as vectors of the malaria parasites. These so-called fever mosquitoes, who prefer regions with high humidity and who splay their abdomens at an angle of roughly 40 from a surface in normal position (Figs. 1 and 2), require the nutrients in the blood of vertebrates in order for their eggs to reach maturation. The males of this genus feed on the sap of plants and are therefore not potential vectors. Eggs, which are able to float using small lateral air chambers, are laid individually at night in batches of 70 100 on the surface of placid waters. The larvae hatch out of these and come to the surface of the water for air. Once there (unlike the Aedes and Culex genera), the larvae lie flat with their abdomens parallel to the surface of the water (Fig. 2). The larvae feed by filtering organic matter. In 13 weeks (depending on temperature) and after three molts (= four larval stages), the larvae reach the pupal stage, during which the pupas are still mobile and come to the surface of the water for air, but no longer feed. The pupal period lasts approximately 35 days and ends with the hatching of a sexually dimorphic male (antenna with many bristles) or a female (antenna with few bristles; Figs. 1 and 2), such that each successive generation takes about 1.53.5 weeks up to 50 days. A female will lay approximately 1,0003,000 eggs in ten clutches during her lifetime. As a result, enormous numbers of potential vectors come into being, especially in tropical regions where the humidity and temperatures provide ideal conditions for year-round development! If there are not enough blood donors near the breeding waters, then the mosquitoes (sometimes swarms of them) are able to travel many kilometers (for example, they can proliferate along coastal zoneswith hotels!). Most Anopheles species are nocturnal, whereby the females bite in the late evening and early morning. Using their olfactory organs, they are able to locate their warm-blooded hosts even in darkness. As

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Fig. 1 a Micrograph of a sugar feeding Anopheles mosquito. b Scanning electron micrograph of the head of a female mosquito

also brings them easily into the vicinity of potential hosts. Thus, the biology of the Anopheles mosquito ensures a high potential for infection for blood parasites, such as malaria parasites, even thoughunlike with other types of parasitesthe malaria parasite is never transmitted to the offspring via the egg (Mehlhorn 2008). As a result, protection against mosquitoes (applying repellent to the skin and using mosquito nets, etc.) cannot be overemphasized. While mosquito control is possible by chemical as well as biological means, a complete eradication of these highly reproductive mosquitoes is likely to remain unattainable. Malaria parasites in humans

vessel feeders, they stick their fine stylet-shaped mandibles directly into blood vessels in the skin. In order to keep the blood from coagulating, they inject an anticoagulant with their saliva, which can cause allergic reactions of its own around the site of the bite (bumps, itching). Mosquitoes are also definitely able to bite through clothing and the gauze of mosquito netting because their mandibles can grow up to 3 mm long. It takes about 24 days for the eggs to develop, making it necessary to feed at this same pace. Since females obtain blood at least ten times from warm-blooded animals (including humans) during sexual maturity, there is a good chance that they will ingest malaria parasites and transmit them within endemic regions. Their flight radius of 23 km/day
Fig. 2 Diagram of the life cycle of vector mosquitoes

Classic human malaria is essentially caused by four species of the genus Plasmodium andwith the exception of a few primateshas no animal reservoir (Mehlhorn 1988). These four species can occur at the same time, which then makes it difficult to reach a diagnosis (see below). In addition, numerous human cases of monkey malaria caused by Plasmodium knowlesi were also recently identified in Asia, even the lethal ones (Thailand, Malaysia, Philippines, etc.); the parasites were transmitted via the bites of Anopheles species, such as Anopheles hackeri, Anopheles lateens, or Anopheles dirus, who feed on both humans and monkeys. Several fatalities have also been described recently (Galinski and Barnwell 2009). In addition, human cases of malaria

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caused by Plasmodium cynomolgi have also occurred. Classic malaria parasites include: Plasmodium vivax (Fig. 3) causes malaria tertiana (benign tertian); it occurs worldwide, mostly in South and Central America, the Middle East, India, rarely in

sub-Saharan Africa, but is not limited to constantly hot climates. Recently, there has been a growing number of reports that P. vivax can cause severe and fatal malaria as well (Anstey et al. 2009). Plasmodium ovale (Fig. 3) causes a tertiana type of malaria, usually focally present not only in tropical

Fig. 3 Light micrograph of red blood cells with the blood stages of the four human malaria species

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regions of Africa but also in Indonesia, the Philippines, Thailand, Vietnamwas it imported there? P. falciparum (Figs. 3 and 4) not only can cause malaria tropica (malign tertian) but can also present with continuous fever or no fever (see below), worldwide in all tropical and subtropical regions. Plasmodium malariae (Fig. 3) causes malaria quartana and it occurs worldwide but only focally present in constantly hot climates.

of the functional genes, while only 18% are species specific corresponding to the relations between humans and the chimpanzee (Carlton et al. 2008a, b; Das et al. 2008; Gardner et al. 2002; Mikkelson et al. 2005). The development of malaria parasites in humans A human is first infected with the injection of usually about 2,000 sporozoites (per bite), which are found in the saliva of the mosquito and grow to approximately 1015m in length with a diameter of only 0.51m. A mere 30 sporozoites is all it takes to cause an infection. Even though they will enter the blood stream and penetrate the hepatocytes (via the endothelial cells and Kupffer cells) within 30 s to 2 min at the most, these sporozoites (Fig. 6) are protected against the hosts defenses by a surface coat of circumsporozoite proteins. Then the exoerythrocytic schi-

The four authentic human types, which differ significantly in both their morphology (diagnostic options) as well as the clinical symptoms that they cause (see below), still have the same basic development cycle (Fig. 5). However, the comparison of the fully sequenced P. vivax and P. falciparum species show that these both species which differ considerably in their pathogenicity share about 82%
Fig. 4 a SEM micrograph of a red blood cell with two P. falciparum schizonts that appear to be pushed through. The penetrated erythrocyte exhibits typical knobs or protrusions on its surface caused by deposits from internal metabolic by-products. b Transmission electron micrograph (TEM) of a red blood cell with two P. falciparum meronts. c TEM of P. falciparum merozoites in a bursting red blood cell

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Fig. 5 Diagram of the life cycle of malaria parasites (according to Mehlhorn 1988). 1 Sporozoites are injected when the mosquito bites. 2 Exoerythrocytic schizogony occurs in the cells of the liver. In some species, some of the sporozoites remain as dormo- or hypnozoites and cause relapses that occur at different intervals, depending on species. 3, 4 Merozoites emerging from hepatic schizonts normally penetrate erythrocytes. In the case of P. vivax, they also appear to penetrate parenchymal cells, though in smaller numbers, and cause subsequent blood parasitemias. 57 Erythrocytic schizogony. Within the erythrocytes merozoites are formed from schizonts over a period of time that varies according to species (see Table 1). This results in typical attacks of fever. 8, 9 One portion of the erythrocytic stages is

transformed into gametocytes, which can survive inside the mosquito. 1013 Formation of gametes. Female (macrogametes, 10) and male gametes (microgametes, 1113) develop within a few minutes after the mosquito feeds. 14 Syngamy, zygote formation. 1517 A motile kinete (= ookinete) grows out of the stationary zygote. 18 The ookinete passes through the peritrophic membrane and penetrates the intestinal epithelial cell of the mosquito. 1921 Formation of sporozoites within oocysts (on the outside of the intestine). 22 Sporozoites in the salivary gland of the mosquito form the surface coat (= become infectious). BM basal lamina, E erythrocyte, IN intestine, LP liver parenchymal cells (hepatocytes), N nucleus, PG pigment, PV parasitophorous vacuoles, SG salivary gland

Parasitol Res (2009) 105:609627 Fig. 6 a, b LM and SEM micrographs of a P. falciparum sporozoite

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zogony = merogony process begins, which will also take place regardless of chemoprophylaxis. The parasite undergoes asexual multiplication, whereby a single dividing schizont can produce varying numbers of merozoites (P. vivax approximately 10,000, P. falciparum approximately 30,000, but P. malariae only 2,000). The time needed for this process depends on the species (Table 1). In the case of malaria parasites, with the exception of P. vivax, only a single generation of merozoites appears to form in the liver (Fig. 5), most of which then penetrate directly into the erythrocytes. However, a small number of merozoites remain in macrophages, etc. Penetrating sporozoites (or even schizonts) also may not develop fully, but instead become the so-called hypnozoites or dormozoites (in P. vivax, P. ovale), which then, after varying periods of time depending on the species, can cause the formation of more merozoites, prompting a new invasion of the erythrocytes. This process is known as a relapse and differs from recrudescence. In the case of the latter one, residual erythrocytic populations reactivate and result in new attacks of fever (for example P. malariae, see below). The merozoites emerging from the schizonts in the liver penetrate the erythrocytes, merely denting their cellular membrane, such that the parasites become lodged inside a
Table 1 Development of the Plasmodium species in humans Species Prepatent period = minimum duration of the exoerythrocytic merogony (days) 8 8 5 1317 Average start for erythrocytic merogony (days) 1317 1317 812 2837

tightly joined parasitophorous vacuole where they can then begin the erythrocytic schizogony process, which is likewise asexual. Individual Plasmodium species prefer to penetrate at different life stages of the erythrocyte. P. falciparum and P. vivax often penetrate during the young stages (reticulocytes). A double invasion of the erythrocytes is also very common in the case of P. falciparum (Fig. 4b). Following penetration (in less than 30 s), the merozoites enter a stage known as the signet ring because of its appearance under a microscope (Fig. 3), in which a spherical parasite has a large central vacuole with peripheral nucleus. Examination under an electron microscope reveals that at this stage (trophozoite), erythrocyte plasma is ingested with a large cytostome (micropore). Through growth and nuclear divisions, schizonts = meronts are formed with a species-specific number of nuclei (Fig. 4b, c). The metabolites of the ingested hemoglobin are stored in crystalline form in vacuoles as so-called pigment. The growth process for the meronts ends with the production of erythrocytic merozoites approximately 1.5m in size, which are released with the pigment and penetrate new erythrocytes after the remains of the host cell burst (Fig. 4c and Fig. 5). The time needed to form these merozoites varies among the different malaria parasites (P. falciparum,

Duration of erythrocytic merogony (h) 48 48 48 72

First occurrence of gametocytes in the blood (days) 1113 2022 2217 2431

Common incubation times (days) 831 1116 527 2037

Retention in the blood (years) 4 4 12 40

P. P. P. P.

vivax ovale falciparum malariae

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P. vivax, P. ovale 48 h, P. malariae 72 h; Table 1). For reasons which are not yet fully understood, the formation of the merozoites within the erythrocytes becomes almost completely synchronized, such that large quantities of pigment and parasites are released simultaneously accompanied by recurring attacks of fever, which are eponymously known as tertian fever. Following a species-specific period of time (Table 1), a portion of the erythrocytic merozoites become gamonts (= gametocytes), while asexual schizogony continues as a parallel process (Fig. 5). Pigment is also stored as a metabolite in these gametocytes, whose shape and coloring vary according to species (see below). The further development of the sexual preliminary stages does not take place until the parasite enters the intestine of the mosquito, whereby the cooling of the blood as it is ingested acts as a stimulant, which has been demonstrated through in vitro studies. The erythrocytic stages of human malaria parasites demonstrate the characteristics shown in Fig. 3 and Tables 2 and 3, which lend themselves to species diagnosis. The development of malaria parasites in the intestines of mosquitoes The female mosquito ingests all blood stages in a blood meal (Fig. 3). However, while the asexual stages are being digested, the gametocytes continue to develop. The formation of the gametes begins a few minutes following the blood meal in the midgut, where the ingested blood is surrounded by a sack (= peritrophic membrane). The male gametocyte divides into four to eight flagellated microgametes, the formation of which has been described in light microscopy studies as exflagellation (Fig. 5). However, the oocyte (= macrogamete) stays behind without undergoing nuclear division and becomes somewhat more spherical. Following fertilization, the zygote elongates to form a motile ookinete, which penetrates the peritrophic mem-

brane, passes through the cytoplasma of the intestinal cells, and settles outside between intestinal epithelium and basal lamina. Here, the ookinete continues to grow and is more correctly termed a zygotokinete, until it becomes an oocyst and seems to form a protective wall around itself (Fig. 7). Asexual multiplication takes place within this oocyst (sporogony), producing thousands of long and slender sporozoites, which are released into the hemocoel when the oocyst bursts and these find their way to the salivary gland of the mosquito. Here, the surface coat is formed that will protect against the human immune system (Fig. 6). The duration of development in the mosquito vector varies depending on the species and is controlled by the ambient temperature. A lower limit of 15C was determined for development in P. vivax and P. malariae; for P. ovale, it was 16C and for P. falciparum, a temperature of at least 1821C is required. The optimal ambient temperature for all malaria parasites has been shown to be 25C. But then differing time periods are required for each species to form infectious sporozoites. In the case of P. vivax, it takes 910 days; for P. falciparum, it takes 1012 days; for P. ovale, it takes 1216 days; and for P. malariae, it takes 1521 days until the process of sporogony is completed within the mosquito. With an ambient temperature of only 20C, these processes can take significantly longer, for example 1617 (P. vivax), 2228 (P.falciparum), and 2025 days (P. malariae). This also lengthens the span of the infection risk in any region following the first occurrences of people falling ill. The amount of malaria infection within a region is measured by various degrees of so-called endemicity. This depends on the so-called entomological inoculation rate (EIR), which results from the number of bites by infectious mosquitoes per person per year within a region. In Africa, the EIR can encompass several thousand bites, but even in highly endemic regions, it is often only 40400.

Table 2 Characteristics of asexual blood stages for human malaria parasites Species Parasite stages in the peripheral blood All All Signet ring only (trophozoites), prior to death: all All Size of the trophozoites 2/5 RBC 2/5 RBC 1/5 RBC Number of schizont nuclei 1224 612 832 Pigment Changes in the cells of the host Greatly enlarged, with Schffners dots Slightly enlarged, ragged surface, Schffners dots Usually none, but sometimes Maurers clefts Usually none

P. vivax P. ovale P. falciparum P. malariae

Yellowish brown Light brown Scattered: light brown; clumped: blackish brown Dark brown

2/3 RBC

612 (8 often arranged in shape of a rosette)

The trophozoites are also known as signet ring stage (because of the peripheral nucleus) RBC red blood cell

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the meronts takes place almost exclusively in the types of erythrocytes, which owing to their surfaces (with typical knob-like protrusions) adhere firmly to the capillary walls within organs such as the liver, spleen, brain, and unfortunately lead to occlusions (thrombi; Knobloch et al. 2003; Mehlhorn et al. 1995). Clinical symptoms of malaria tropica In the case of this most severe form of malaria (Fig. 3), which is often fatalif left untreatedthe following symptoms (usually in combinations) are the most common symptoms among infected persons: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. Severe fever (99100%) Headache (84%) Chills (81%) Splenomegaly (69%) Anemia (68%) Outbreaks of sweating (67%) Nausea (39%) Vomiting (39%) Arthralgia (39%) Diarrhea (18%) Coughing (16%) Abdominal pain (16%)

Fig. 7 TEM micrograph of a Plasmodium oocyst on the outside of the mosquito intestine

Diagnosing human malaria parasites Diagnosing human malaria parasites is relatively simple based on the morphology of the blood stages (Fig. 3; Tables 2 and 3) with the exception of P. falciparum. Due to the fact that a misdiagnosis is possible for this most dangerous parasite, it is imperative to exercise great caution. Generally, Giemsa-stained blood smears and thick blood films are sufficient. Both procedures should always be used simultaneously because with double infections, the P. falciparum parasites can be hidden behind a large number of merozoites from another species. While the time at which blood is withdrawn does not matter for the other malaria parasites, in suspected cases of P. falciparum (see clinical aspects), it is imperative that blood be tested every 6 h (with continuous fever) or at the end of a significant attack of fever (with tertian fever). This is due to the fact that in the case of P. falciparum, the maturation process for

In lab tests, serious deviations were found for the following parameters:
Thrombocytopenia (standard >150,000l) LDH (standard <240 U/l) Leukocytopenia (standard 5,000/l) Anemia (standard: Hb females >12 g/dl, males 14 g/dl) 5. ALT (standard: female <19 U/l, male <23 U/l) 6. Gamma-GT (standard: female <18 U/l, male <28 U/l) 7. AP (standard <190 U/l) 1. 2. 3. 4. 100% of patients 74% of patients 68% of patients 68% of patients 48% of patients 48% of patients 48% of patients

Table 3 Characteristics of the sexual blood stages of human malaria parasites Species P. vivax P. ovale P. falciparum P. malariae Shape Spherical/ ovoid Spherical/ ovoid Sickleor bananashaped Spherical/ ovoid Microgamont 10m; red, eccentric nucleus; light blue/ pink plasma; pigment in the form of fine grains 9m; similar to P. vivax 911m, large and diffuse nucleus, pink plasma, diffusely scattered pigment 7m; similar to P. vivax Macrogamont 11 mm, small, dark red nucleus; blue plasma; lots of pigment 9m; similar to P. vivax 1214m, central and red nucleus, blue-to-purple plasma, pigment centered around the nucleus 7m, similar to P. vivax

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There is a risk of multiple organ failure, which is ultimately the cause of death in most cases of malaria tropica. In addition to these measurable symptoms, more complicated cases of cerebral malaria tropica may also include numerous other symptoms, such as strabismus, miosis, hallucinations, disorientation, nystagmus, ataxia, psychoses, spasms, tremors, hemiparesis, weakness of the facial muscles, hemoglobulinuria, jaundice, acidosis, etc. (Knobloch et al. 2003; Mehlhorn et al. 1995). With parasitemia levels at or above 40% of red blood cells, the prognosis for surviving malaria tropica is extremely poor, despite treatment. In the case of the primary, nonimmediately lifethreatening species of malaria (Malaria tertiana, Malaria quartana) direct symptoms include fever every 48 or 72 h, respectively. However, in cases of double infections, the bouts of fever can overlap and thus appear atypical. Seldom are more than 23% of the red blood cells infected with these species. The incubation times for both malaria species can last a very long time (weeks to months), such that a possible link with a previous trip to the tropics is often overlooked (Mehlhorn 2008; Mehlhorn et al. 1995). Pathogenicity factors for severe = complicated malaria Severe symptoms and cerebral infection may occur, especially in the case of P. falciparum infections and recently in the case of P. vivax infections as well (Anstey et al. 2009). The degree of severity of certain factors ranges from the known spectrum of asymptomatic progression to complications resulting in death. Thus, the following factors have an influence on the progression of the disease: & & & & Reduced blood flow due to the adhesion of infected erythrocytes (Fig. 4a) to vascular walls or through the formation of thrombi in narrow blood vessels Upregulation in the development of adhesion molecules due to cytokines Increased cytokine induction as a result of the release of toxic by-products from the parasites Secretion of nitric oxide from endothelial cells and various other sources

Combating malaria History of malaria treatment from the European perspective Since the dawn of human history, it is likely that attempts have been made to treat malaria with herbs and other natural remedies because malaria parasites were present in the Rift Valley, the cradle of humanity, from the very beginning.

The oldest known record of a specific malaria treatment using herbal extracts comes from China in the year 340 A.D. In the Handbook of Prescriptions for Emergency Treatments, Ge Hong describes a tea made from a handful of mugwort herb, which is effective in reducing fever (Klayman 1985). The fever-reducing effect of mugwort herb (genus Artemisia) is based on the plasmodicidal properties of the ingredient artemisinin, which is synthetically produced today and to which almost no resistances exist to date (Noedl et al. 2008). However, just recently (June 2009), a retarded activity of Artemisinin in Cambodia was announced even by BBC London after being shown already by Noedl et al. (2008). In Europe, the healthful effect of mugwort was unknown up until the twentieth century, even though the need for malaria medications was high because of the presence of malaria in nearly every European country. Nowadays, however, European malaria and its history have mostly faded into obscurity. Originally, nearly all the countries north of the Alps and all the way to Scandinavia were malaria-endemic regions. In 1735, Linnaeus wrote about malaria in Sweden in his medical doctorate dissertation: here there is no illness that is more native nearly all the students in Uppsala are affected by it (Uppsala-Universitet 2008). In 1946, Reiner Mller reported in his textbook on medical microbiology: Following the Napoleonic Wars, many of the lowlands in Germany were tainted, and namely stricken with Tropica as well; probably due to carriers of gametes returning home from southern theaters of war. Very virulent in the Rhineland in 1826/27 near Duisburg, Grevenbroich, Jlich and Aachen. At the Jlich fort about a quarter of the occupants (soldiers) fell ill as late as 1835. On the west coast of Schleswig Holstein 28% of the residents fell ill in 1826/27 (Mller 1946). Overall, European malaria was a widespread parasitosis, which, in addition to its endemic prevalence, continued to exhibit epidemic-scale outbreaks and had considerable social and economic significance. While the non-tropica forms of malaria resulted in few deaths and chiefly caused morbidity due to periodically recurrent attacks of fever and limited economic achievement potential among those affected, malaria tropica claimed countless lives, wherever it appeared. The history of the spread of malaria tropica in ancient Italy can be traced to about 750 B.C. It is believed that the parasite was introduced at that point in time from Africa (Sallares et al. 2004). Malaria tropica was decisive in determining settlement patterns throughout the Mediterranean region for more than two millenia. Bruce-Chwatt and de Zulueta write tellingly: Medieval Italy was no longer the powerful and densely settled land that it had once been during the classical period. Cities shrank and sometimes disappeared, leaving behind ruins for the modern traveler to ponder in amazement. Expansive areas of fertile land were abandoned (Bruce-Chwatt and de Zulueta 1980).

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An effective drug for the treatment of malaria was not available to people, such that malaria was a totally catastrophic disease among the masses up until the seventeenth century. This changed around the year 1633, with the emergence of imported Cinchona tree bark, which would turn out to be the first specifically effective antimalarial agent available in Europe (Fig. 8). According to the surviving legend, Linnaeus (17011778) supposedly named the tree (genus Cinchona) after the second wife of the Spanish viceroy of Peru, Francesca Riverade Chinchon, who arrived in Peru in 1629 and was cured by her native Indian servant with the bark of the tree in 1638. This led later to the catchy term china bark. Cinchona alkaloids occur naturally in the bark of Cinchona and Remijia trees, which naturally occur throughout Venezuela and Bolivia. It was the Spanish Jesuits who learned of the fever-reducing effects of the bark extracts from the indigenous people of South and Central America and they [the Jesuits] exported it to Europe (Lee 2002). In Europe, the bark was sold under the names Peruvian bark, china bark, or fever bark, as well as Jesuits powder or Countesss powder.
Quinine This bitter substance is contained in the bark (515%) of approximately 6-year-old trees known as Cinchona. Cinchona pubescens, Cinchona succirubros, Cinchona officinalis, Cinchona ledgeriana, etc. belong to the Rubiaceae family of plants, which grow at 8003,000 m in the Andes (Fig. 8) and are later exported to Asia. The name comes from two sources. Linnaeus named the genus Cinchona, after the name of a Spanish vicereine in Peru. The Incas the indigenous people of Perucalled the bark of this tree quina quina = bark of the bark. These names then underwent an onomatopoeic shift over the course of the centuries, eventually giving rise to the term china bark. Today, the alkaloid quinine is instrumental in saving the lives of those affected by resistant cerebral malaria tropica.

The English chemist Robert Tabor (16421681) played a very important role in the distribution of the Cinchona bark. He performed experiments with it and developed a special technique to extract the quinine it contains; he also propagated its specific antimalarial properties (Lee 2002; Siegel and Poynter 1962). In Italy, the personal physician of the Duke of Modena, Francesco Torti (16581741), is credited with first describing malaria as an illness and specifying Cinchona bark as a therapeutic agent. Ultimately, the Cinchona tree received its official scientific genus name Cinchona through none other than the famous Swedish naturalist Linnaeus (17011778) in the year 1742 (Linnaeus 1748); he had earned his medical doctorate degree 7 years earlier after writing his doctoral thesis on Swedish malaria; as director of the Hortus Botanicus Amsterdam, he also established the taxonomy of Linnaeus for classifying animals and plants, which is still in use to this day (Bruce-Chwatt 1988). Cinchona bark

contains the active ingredient quinine, which is still an indispensablesynthetically manufacturedmalaria medication to this day. After its discovery, Cinchona bark was a scarce and coveted commodity, which was only available initially as an import from South America. In his search for local alternatives, the British physician Samuel James (1763 1831) discovered the medicinal effect of salicylic acid in the year 1792. He administered his malaria patients with preparations made with willow bark (Salix caprea), which had a bitter taste similar to that of the Cinchona bark and was believed to contain the same active ingredients. James actually observed a fever-reducing effect (Kuhnert 2000; Pierpoint 2007). It was discovered only later that the feverreducing effect of the willow bark is not based on the destruction of malaria parasites but rather on the effect of the salicylic acid it contains, which is the primary ingredient in aspirin; while salicylic acid symptomatically reduces fever, it is not able to destroy malaria parasites. The sharp increase in worldwide demand for Cinchona bark led to a series of expeditions to the New World of South and Central America, which was little known at that time. The German botanist Justus Karl Hasskarl (1811 1894) is believed to have been one of the first explorers to bring Cinchona tree cuttings and seeds back to Europe from Peru in 1854 at the behest of his Dutch clients (Dnschede 1971). A short time thereafter, Charles Ledger (1818 1906), an English commercial traveler, succeeded in importing 14 English pounds of Cinchona seeds to England. Following unsuccessful negotiations with the British government in 1865, these seeds were also ultimately sold to the Dutch (Bruce-Chwatt 1988), who were having massive problems with malaria in their colonies in the East Indies. Thus, it was the Dutch who were ultimately successful in cultivating Cinchona trees in their colony of Java and it was discovered that the bark of these trees contains a remarkably high 1113% of the active ingredient quinine. In honor of Charles Ledger, the trees later received the scientific name Cinchona ledgeriana. However, there is a whole series of related species. By 1877, the Javan plantations comprised some 20,000 Cinchona trees, and the prices for their bark hit all-time highs in Amsterdam. While at first the quinine market was being served by the smaller dealers, ultimately the lucrative production of quinine was almost completely in the hands of a few large producers by the end of the nineteenth century; from 1918 onward, it was then exclusively controlled by the Dutch (Bruce-Chwatt 1988). It was not until 1929 that quinine was completely synthesized by Paul Rabe (Rabe 1932; Smith and Williams 2008). An important milestone in the history of malaria and its prevention was the discovery of the parasite by Charles Louis Alphonse Laveran (18451922), a physician at the

620 Fig. 8 Image of the bark from different species of Cinchona trees

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French military hospital in Constantine (Algeria); in 1880, he noticed spherical bodies in the blood of malaria patients (Bruce-Chwatt and De Zulueta 1980), for which he was to receive a Nobel Prize in 1907. By 1920, the development cycle of the Plasmodium parasite in the Anopheles mosquito was able to be completely explained. Between 1940 and 1950, the liver stages of the parasite were unequivocally identified in humans as well. The targeted development of medications had not been possible until these processes were fully understood. The birth of synthetic antimalarial agents Due to the Dutch monopoly on quinine and spurred by further developments in chemistry, an intensive search for synthetic antimalarial medications began around 1900. The chemotherapeutic research laboratories of the I.G. Farbenindustrie AG in Wuppertal-Elberfeld (Germany) were especially active in this field. At first, however, it was only possible to successfully synthesize less potent substances. The lack of a suitable malaria test model was especially limiting with respect to the full-scale systematic testing of new substances. In 1925, Walter Rhl (18841923) established the avian malaria model, which had already been described for the first time in 1911 by Phokion Kopanaris (Kopanaris 1911). Using this model, scientists

ultimately succeeded in discovering the antimalarial effects of plasmochin and atebrine, which entered the market in 1927 and 1932, respectively. With the advent of these two substances, synthetic antimalarial agents superior to quinine were finally available to be produced and these were soon in wide use in the period that followed (Fig. 9). In 1932, the Dutch-East Indian quinine monopoly, alarmed by the success of the German scientists, imposed an embargo on the export of the Java sparrow, upon which the avian malaria test model was based (Dnschede 1971). In the event, however, lab testing was only temporarily halted and by 1934 Hans Andersag (19021955; Fig. 10) was able to successfully synthesize Resochin, which would later turn out to be the most important antimalarial agent to date (Figs. 11 and 12). Resochin (chloroquine) The history of resochin began in June 1934 in the chemotherapeutic research laboratories at I.G. Farbenindustrie AG in Wuppertal-Elberfeld, where Hans Andersag, in the search for new antimalarial agents, synthesized 4-aminoquinolines (Fig. 10). One of the first derivatives to be tested, 2.4dihydroxybenzoic acid, was shown to be especially effective in the avian malaria model and received the name resochin (from the German, RESOchinat des 4-AminoCHINolin).

Parasitol Res (2009) 105:609627

621

Fig. 9 Structural formulae for quinine, plasmochin, atebrine, and resochin. In quinine, the quinoline nucleus is connected with the basic side chain via a carbon bridge, while the bridge in plasmochin is composed of nitrogen. Atebrine is similar to quinine and characterized

by a basic side chain in the gamma position on the pyridine nucleus. Using this basic structure, Andersag and his two colleagues, Breitner and Jung, were able to develop resochin (quinolones alkylated under basic conditions)

Walther Kikuth (18961968), who performed the in vivo testing on the avian model, judged the substance to be similar to atebrine (Fig. 9) in efficacy, but considered it to be somewhat more toxic (Coatney 1963). Franz Sioli (1882 1949) conducted the first resochin trials on humans at the psychiatric hospital in Dsseldorf using paralytic patients, who had been treated with inoculated vivax malaria (German: Vivax-Impfmalaria), which was an established therapy in those days; he observed that the efficacy of the substance was similar to that of atebrine (Fig. 9), but was too toxic for use in humans (Coatney 1963; Dnschede 1971). As a result, this substance was

Fig. 10 Portrait of the discoverer of Resochin: Hans Andersag (19021955)

not subjected to any further clinical study and the scientists at Elberfelde concentrated their efforts on the development of a methylated resochin, later named sontochin (Coatney 1963). In 1941, resochin and sontochin were licensed to Winthrop Chemical Company in the USA (Coatney 1963), where no further clinical advances were made initially. It was not until 1943 when the US government set up the Board for the Coordination of Malarial Studies with the goal of developing new antimalarial medications that the promising effects of resochin were rediscovered, though this occurred via the unintentionally circuitous route of a parallel development of the substance SN-7619 (Coatney 1963). The test substance SN-7619 then received the name chloroquine, but it was identical to the previously established resochin. From 1946 onward, chloroquine became available for use in the field of medicine (Haberkorn 1988). Numerous clinical studies were initiated with chloroquine manufactured by Winthrop and a comprehensive understanding soon emerged that the drug was reliably clinically effective and well tolerated. Starting in 19505 years after the end of the warBayer AG in Leverkusen (Germany) began manufacturing and distributing chloroquine again (Haberkorn 1988). The original chemical name resochin became the trade name Resochin and chloroquine became the chemical name for the substance. News of its therapeutic success spread rapidly and a couple of years after its introduction, chloroquine had come into widespread and successful use throughout almost every part of the world where malaria was present (for example between 1960 and 1964, it was one of the most widely sold medications). In addition, its long serum half-life (BayerHealthcare 2008) made it ideal for use as a prophylactic and for the prevention of reinfection in successfully treated patients. In general, chloroquine is considered to be a well-tolerated medication, which warrants its widespread use

622

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Fig. 11 Monthly report (June 1934) from Andersags laboratory with the structural formula for Resochin, Circle = Resochin

as a treatment and a prophylactic. The number of people who owe their lives to chloroquine has not been documented. It is believed to be in the hundreds of millions, though this is merely an extremely rough estimate. Nevertheless, some 250 million people are infected with malaria each year, according to current statistics, while twice that number are at risk for infection; this provides a clear picture of the dimension of the malaria problem (WHO 2008). Chloroquine gained special significance as part of the WHO Malaria Eradication Program begun in 1955 (Greenwood et al. 2008). The plan was to achieve vector control using insecticides, especially dichlorodiphenyltrichloroethane (DDT). At the same time, all infected persons were to be located and treated in order to destroy the reservoir for the parasites that exists within humans. Chloroquine was the most important therapeutic agent used for the treatment of the masses (Greenwood et al. 2008). The eradication program met with a few successes: by 1982, the WHO was able to declare 24 countries to be endemically malaria-free, including the countries of North America and all of Europe. The key to this success was the combined use of the most diverse methods available, for example monitoring as well (BruceChwatt and De Zulueta 1980), which placed high demands on

the infrastructure of each country. In this respect, countries with a lower level of development were at a disadvantage. However, the problem of African malaria was so intractable that the WHO was never able to achieve eradication there. However, the actual geographic distribution of malaria is currently far different from the distribution pattern that existed before the eradication program because nearly every malaria-free region has been able to maintain that status. Effect of chloroquine (Resochin) The method of action of chloroquine is based on the disruption of the detoxification function in malaria parasites. Plasmodium trophozoites take up large amounts of hemoglobin into their digestion vacuoles during their intraerythrocytic cycle (a and b in Fig. 4) and eliminate the degradation by-product hematin, which is toxic to them, by means of polymerization and binding of inert hemozoin crystals and presumably through an additional degradation process facilitated by glutathione. Chloroquine binds to hematin gamma-oxodimers and is deposited on the surface of the hemozoin crystals. This ultimately destroys the parasites (Wellems and Plowe 2001).

Parasitol Res (2009) 105:609627 Fig. 12 Patent issue notification for the publication of the patent for the Resochin active ingredient patent (683692)

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Chloroquine resistance Starting in 1957, the first case reports of chloroquine resistance began cropping up (Table 4) for P. falciparum malaria from places as distinct from one another as the ThailandCambodia border region and Columbia (Talisuna et al. 2004). Chloroquine resistance has been spreading across the globe ever since. By 1980, nearly all of the endemic regions in South America were affected and the same was true for corresponding countries in Asia by 1989. Chloroquine resistance occurred for the first time in Africa around 1978 and spread rapidly thereafter across the entire continent in the same way it was observed to spread elsewhere (Talisuna et al. 2004).

The reasons for the emergence of this resistance are many and they are certain to be due to a combination of different factors. Mutations seem to occur with a combination of the following factors (Knobloch et al. 2003): 1. Uncontrolled long-term regimens (for preventive treatment) often associated with low doses of the active ingredient 2. Travel activity by the person being treated (resulting in the spread of nonsensitive strains) 3. Frequent feeding by Anopheles mosquitoes from different hosts, resulting in the local spread of resistant parasites 4. Simultaneous emergence of different resistance genes

624 Table 4 Characteristics of the stages of chloroquine resistance (RIRIII) Stage Sensitive RI RII RIII Parasitological parameters following treatment Blood remains free of parasites Following parasite-free interval of 728 days parasite recrudescence Reduction in the parasite count by more than 75% after 48 h, but the parasites are still visible for at least 7 days Reduction in the parasite count by less than 75% after 48 h Clinical characteristics

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Clinically cured (ACR) Clinical symptoms return Clinical symptoms remain (ETF) Clinically severe symptoms due to treatment failure (LTF)

ACR adequate clinical response, ETF early clinical treatment failure, LTF late treatment failure

5. The mutated parasites appear in the blood of the host in greater numbers than the sensitive parasites. This results in a higher parasite load within the vector. 6. The chaos of wars and the lack of malaria controls also result in the spread of these kinds of resistant strains as soon as they emerge.

Unlike P. falciparum, in many countries, the P. malariae and P. ovale species are still almost completely sensitive to chloroquine. The same is true for P. vivax in most of the regions where it occurs. Thus, chloroquine is still an invaluable tool for prevention and treatment in malaria regions where P. falciparum is not endemic. The emergence of chloroquine resistance had a dramatic impact on malaria mortality rates in many countries. It has been shown that the malaria mortality rates in many regions of West Africa increased two- to eightfold with the emergence of chloroquine resistance (Trape et al. 1998). Infants and children were especially affected by this increase in mortality. It was able to be proven that the accumulation of chloroquine decreases in the digestion vacuole of chloroquine-resistant Plasmodium strains (Wellems and Plowe 2001), which indicates resistance mutations in transport proteins. The pfcrt gene is localized to chromosome 7 and coded for P. falciparum chloroquine-resistant transporter protein (PfCRT), a transmembrane protein in the digestion vacuoles of P. falciparum (a and b in Fig. 4). Several mutations of this gene have since been identified that result in a resistance to chloroquine. There are wellknown mutations of the pfcrt gene, for example, from numerous regions in Africa, Asia, and South America (Wellems et al. 2001). Similarly associated with chloroquine resistance are mutations of the pfmdr1-gene (= P. falciparum multidrug resistance gene 1) on chromosome 5, which is coded for the Pgh1 gene product. Mutations of this gene locus are found in the chloroquine resistances of P. falciparum strains, for example, in Mali, Gambia, Sudan, Thailand, or Brazil. In 1993, Malawi was the first African country to switch to sulfadoxinepyrimethamine as the drug of first choice for

the treatment and prevention of malaria because of the high resistance rates to chloroquine (Laufer et al. 2006). Chloroquine is practically no longer used in Malawi, such that the chloroquine selection pressure on Plasmodium parasites dropped sharply. Eight years after the switch from chloroquine to sulfadoxinepyrimethamine (Kublin et al. 2001) studied the persistence of the pfcrt T76 mutation, which had previously been widespread in Malawi, and they made a shocking discovery: The mutation had disappeared from nearly every isolate they analyzed. However, since there was still some uncertainty as to whether chloroquine would now be sufficiently clinically effective, a prospective randomized study was conducted on 210 patients with uncomplicated malaria tropica to compare chloroquine versus sulfadoxinepyrimethamine. In fact, the therapeutic efficacy for chloroquine was shown to be 99%, while sulfadoxinepyrimethamine was only effective in just 21% of the cases (Laufer et al. 2006). This study proved that it was possible to achieve nearly complete chloroquine sensitivity just 8 years after the total discontinuation of chloroquine use in Malawi. Similar observations on the restoration of chloroquine sensitivity for P. falciparum have since been made as well in Tanzania, South Africa, China, and Thailand (Read and Huijben 2009). Apparently, Malawi was not an isolated case. These observations gave rise to the hope that it might be possible to restore chloroquine sensitivity in other regions by means of targeted drug cycling, thus ensuring that chloroquine will once again become available as a reliable and inexpensive antiamalarial medication for the treatment of malaria tropica in the future. It remains unclear how effective the implementation of such a drug cycling concept will ultimately be and whether it can be applied to other antimalarial medications as well. But one thing is for sure, chloroquineonce againis playing a pioneering role. Characteristics and dosing instructions for Resochin (chloroquine) Resochin (Figs. 9, 11, 12, 13a and 13b) exhibits the characteristics shown in Table 5.

Parasitol Res (2009) 105:609627 Fig. 13 a, b Earlier packages of Resochin. c Historical advertisement for Resochin

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Table 5 Characteristics and dosing instructions for Resochin Active ingredient Chloroquine N-(7-chloroquinolin-4-yl)-N,N-diethyl-pentane-1,4-diamine Latin: Chloroquinum C 18 H 26 ClN3 54-05-7 50-63-5 (diphosphate) 2719 DB 00608 PO 1 BA 01 white or almost white hygroscopic crystalline powder (diphosphate) Antiprotozoals Resochin, Chlorochin, Nivaquin, Weimerquin 319.88 pmol1 289C 10.1 Water 10.6 mg/l at 25C Not readily soluble in ethanol and methanol (diphosphate) 330 mg/kg body weight in rats (oral administration) Preventive treatment Adults500 mg chloroquine phosphate=2 tablets (250 mg each) per week Children8.1 mg chloroquine phosphate/kg body weight per week (maximum dose 500 mg/week) Duration1 week prior to 4 weeks following a stay in a malaria region Emergency treatment (adults and children) Begin treatment with 16 mg chloroquine phosphate/kg; after 6 h take 8 mg chloroquine phosphate/kg; for the next 23 days take 8 mg chloroquine phosphate/kg once daily; microscopy analysis is absolutely necessary (smears/thick blood films)

Chemical formula CAS number Pubchem Drug bank ATC code Appearance Class Proprietary names Molar mass Melting point pKa value Solubility LD 50 (Crouzette et al. 2005) Dosing instructions (where sensitivity still exists) according the German summary of product characteristics for Resochin tablets, 250 mg and Resochin junior tablets, 81 mg (BayerHealthcare 2008)

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Is the threat from malaria on the rise in this era of global warming? Since the end of the last ice age approximately 13,000 years agowhen todays North Sea was dry land and a person could have hiked from Germany to England or from Borneo to Australiathe sea levels have risen by about 90 m. The thawing of the glaciers resulted in dramatic changes in the climate everywhere. Large tropical regions expanded and malaria parasites that had existed for more than 90 million years found plenty of Anopheles mosquitoes to act as vectors. But the final hosts, humans (arising about 4.5 million years B.C.), were far fewer than they are today, such that the spread of malaria parasites must certainly have been a seldom occurrence. With the overpopulation of the planet by humans, the situation has now changed completely. Nowadays, there are millions of carriers of malaria parasites poised to be bitten by mosquitoes. Thus, the reliable assumption that malaria will spread in the foreseeable future is not based on the fact that local temperatures are predicted to increase by 5C, but instead on the seemingly unstoppable growth of the human population; more importantly, this expanding human population can only be expected to receive the most basic level of medical care. Although many a media-savvy prophet of doomsday scenarios has already predicted that Germany will become a malaria region, this is not going to come to pass within the next 200 years (at least if it can be assumed that the quality of medical care will remain unchanged). But it is true that over the last thousand years, Germany has repeatedly experienced outbreaks of malaria when it was successfully transmitted by indigenous Anopheles species from people who had become infected with the disease. These people, who had journeyed to subtropical and tropical regions to fight wars, travel the seas, buy and sell goods, etc., came into contact with the parasite and then brought it with them to Germany. Nowadays, however, medical advancements are standing in the way of an outbreak or a reestablishment of endemic malariaeven if the temperature should rise by 5C and even though it is becoming increasingly difficult to combat malaria with medications. This prognosis is supported by the fact that there is no endemic malaria in northern Australia, which is much warmer on average. Nevertheless, in those hot and humid tropical regions with steady population growth and declining medical care, malariatogether with viral epidemicswill become a serious problem that will decimate the population. This means that the temperate countries of todayeven if temperatures rise by 5Cwill have problems other than malaria. Conversely, the overpopulated areas will suffer tremendously from malaria unless an affordable vaccine can indeed be developed.

Prognosis A vaccine against malaria that is effectivebut above all affordableis not going to be found within the foreseeable future, despite the grandiose promises made by many since 1970. It is also true that resistances have emerged against a whole series of antimalarial medications (even the newer ones!), including Resochin as well, which is now in its 75th year. But experience has shown that if these types of medications are discontinued for long periods, they can achieve their former efficacy all over again. This has also been shown for Resochin and is caused for hope, especially since the number of emerging resistances to the alternative medications is on the rise. Thus, we must continue to responsibly manage the dose levels and treatment timeframes for those medications that are still available, well tolerated, and more or less affordable, such as Resochin (chloroquine), which has already had its heyday and just might enjoy another one (Fig. 13).
Acknowledgements We thank BAYER business services, corporate history & archives for the historical images 1013.

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