532

MalariaNICHOLAS J. WHITE

43 

IntroductionMalaria is the most important parasitic disease of man. The malaria parasite is a mosquito-transmitted protozoan. Plasmo-dia are sporozoan parasites of red blood cells transmitted to animals (mammals, birds, reptiles) by the bites of mosquitoes. Protozoan parasites of the phylum Apicomplexa contain three genetic elements: the nuclear and mitochondrial genomes

characteristic of virtually all eukaryotic cells and a 35-kilobase (kb) circular extrachromosomal DNA. This encodes a vestigial plastid (the apicoplast) that is an evolutionary homologue of the chloroplasts of plant and algal cells. Four species of the genus Plasmodium infect humans preferentially and the simian parasite P. knowlesi is an important cause of human malaria in parts of South-east Asia. Occasional infections with other errant primate malarias may also occur.1 Parasites very similar to those infecting humans are found in African great apes. The indi-vidual characteristics of the four species of human malaria parasites are shown in Table 43.1. Almost all deaths and severe disease are caused by P. falciparum. In phylogenetic terms, this parasite is closest to the avian malarias (P. lophurae, P. gallina-ceum), but it is now clear that P. falciparum has co-evolved with its human host through several evolutionary bottlenecks and was not a recent acquisition from birds as once thought. The three ‘benign’ malarias, P. vivax, P. ovale and P. malariae, all lie closer together on the evolutionary tree near the other primate malarias. It has recently been discovered that ‘P. ovale’ comprises two sympatric non-recombining parasite species tentatively termed P. ovale wallikeri and P. ovale curtisi.2 Severe disease with these species is relatively unusual, although occasionally patients will die from rupture of an enlarged spleen and they reduce birth weight which predisposes to neonatal death. Full genome sequences are now available for hundreds of P. falciparum and P. vivax isolates and many of the other Plasmodia. The remark-ably AT-rich P. falciparum genome is approximately 23 Mb in size and encodes about 5300 genes on 14 chromosomes.

HistoryMalaria, or ague as it was commonly known, has been described since antiquity. Hippocrates is usually credited with the first clear description among occidental writers: In Epidemics, he distinguished different patterns of fever and in his Aphorisms, he describes the regular paroxysms of intermittent fever. In Europe, seasonal periodic fevers were particularly common in marshy areas and were frequently referred to as ‘paludial’ (L. palus, marshy ground; Fr. paludisme). In the early nineteenth century miasmatic influences were believed to cause a variety of diseases. Malaria was thought by Italian writers to be caused by the offensive vapours emanating from the Tiberian marshes. The word ‘malaria’ comes from the Italian and means literally ‘bad air’. Indeed the cause of the seasonal periodic fevers was a continuous source of debate until the late nineteenth century. The work of Meckel, Virchow and Frerichs had established that the dark malaria pigment (mistakenly thought to be melanin) observed in the blood of some patients with periodic fever resulted from the destruction of red blood corpuscles. This same pigment caused the characteristic grey discoloration of the internal organs in patients dying from this disease. In the

KEY POINTS

• Malaria is a protozoan infection of red blood cells trans-mitted by the bite of a blood-feeding female anopheline mosquito.

• It is the most important parasitic disease of humans and often the most common cause of fever in the tropics.

• Human malaria infections are caused by Plasmodium falciparum, P. vivax, P. malariae, P. ovale and also by the simian parasite P. knowlesi.

• Malaria is prevalent across the tropical world. In Africa, P. falciparum predominates, whereas in many parts of Asia and the Americas P. vivax is more common.

• Malaria is diagnosed by microscopy of suitably stained thick and thin blood smears or by rapid diagnostic tests, which detect parasite antigens in the blood.

• The clinical manifestations of malaria are fever, anaemia and splenomegaly. Most deaths result from P. falci-parum infections which may cause coma (cerebral malaria), acidosis, severe anaemia, renal dysfunction and pulmonary oedema.

• Treatment of uncomplicated malaria is with artemisinin combination treatments which combine the rapidly acting and rapidly eliminated artemisinin compounds with more slowly eliminated antimalarial drugs. P. vivax, P. malariae, P. ovale and P. knowlesi may also be treated with chloroquine.

• P. vivax and P. ovale malaria also require treatment with primaquine to eliminate the persistent liver forms (hyp-nozoites) which cause relapse of the infection.

• The key elements of malaria control are effective drug treatment, deployment of insecticide-treated bed nets and where appropriate indoor residual insecticide spray-ing, supplanted in some areas by intermittent preven-tive treatments and mass chemoprevention given to all the target population.

• The main threats to malaria control are increasing anti-malarial drug resistance and increasing insecticide resis-tance. There is currently no available malaria vaccine.

SECTION 9 Protozoan Infections

43  Malaria 533

neurosyphilis. This became standard practice throughout the world until the introduction of penicillin 30 years later. Malaria became the most studied infection of humans. Overall, at a time when GPI accounted for 10% of all mental hospital inpatients in Europe, malaria therapy gave approximately 30% of patients a full and 20%, a partial remission of this debilitating and ulti-mately lethal infection.

Until the nineteenth century, malaria was found in northern Europe, North America and Russia – and transmission in parts of southern Europe was intense. Since then it has been eradi-cated from these areas and the number of cases in the Middle East, China and parts of Asia and South America has fallen, but elsewhere in the tropics there was a resurgence of the disease. Between 1970 and 2000 the number of cases world-wide and the number of deaths steadily increased. This rising death toll was not a result of failing health systems as the number of deaths from many other infectious diseases fell. It resulted from increasing resistance of the anopheline vector to insecticides and of the parasite to the antimalarial drugs that were deployed.

Life CyclePRE-ERYTHROCYTIC DEVELOPMENT

Infection with human malaria begins when the feeding female anopheline mosquito inoculates plasmodial sporozoites at the time of feeding. The small motile sporozoites are injected during the phase of probing as the mosquito searches for a vascular space before aspirating blood. In most cases, relatively few sporozoites are injected (approx. 8–15), but up to 100 may be introduced in some instances. Most sporozoites come from the larger salivary ducts and represent only a small fraction of the total number in the salivary gland. After injection, they enter the circulation, either directly or via lymph channels (approx. 20%) and rapidly target the hepatic parenchymal cells. Within approximately 45 minutes of the bite all sporozoites have either entered the hepatocytes or have been cleared. Each sporozoite bores into the hepatocyte and there begins a phase of asexual reproduction. This stage lasts on average between 5.5 (P. falci-parum) and 15 days (P. malariae) before the hepatic schizont ruptures to release merozoites into the bloodstream (Table 43.2). In some instances, the primary incubation period can be much longer. In P. vivax and P. ovale infections a proportion of the intrahepatic parasites do not develop, but instead rest inert as sleeping forms or ‘hypnozoites’, to awaken weeks or months later and cause the relapses which characterize infections with these two species.3 During the pre-erythrocytic or hepatic phase

1870s, medicine slowly moved towards the germ theory of disease, following the pioneering work of Koch. In 1879, Edwin Klebs and Corrado Tommasi-Crudelli reported the identifica-tion of a bacterial cause of malaria. Recovery of the ‘organism’, Bacillus malariae, from patients with malaria was confirmed by several influential Italian physicians and pathologists – and similar reports began to appear in the USA. It was not surpris-ing, therefore, that the report of a French Army surgeon working in Algeria, claiming that malaria was caused by a parasite, was treated initially with some scepticism. On 20 October 1880 (in a later publication he gives the date as 6 November), Charles Louis Alphonse Laveran was examining the fresh blood of a patient with ague and observed moving bodies (he was prob-ably watching gametocyte exflagellation), which he surmised correctly were parasites of the red blood cells. The transmissibil-ity of the infection in blood was proved 4 years later by Ger-hardt, but the route of natural infection was not discovered until the next decade. Following the suggestion of Patrick Manson, a young Scottish physician in the Indian Medical Service, Ronald Ross, began to investigate the possibility that malaria could be transmitted by mosquitoes. In 1897, after many months of failure, he reported the presence of pigmented bodies in the gut of a certain species of brown ‘dapple winged’ mosquito fed on patients with malaria. He speculated that these might represent the parasite stage in the mosquito (he was in fact describing the oocysts) but, because of difficulties in obtain-ing these ‘unusual’ mosquitoes and his transfer to Calcutta, he was unable to characterize the complete life cycle, i.e. transmis-sion from human to mosquito to human. After many years of study, Ross finally proved the existence of the complete life cycle involving a mosquito in the malaria of canaries. He identified the anopheles mosquito as the vector of human malaria, although by the time Ross finally had the opportunity to dem-onstrate Plasmodium falciparum sporogony in anopheline mos-quitoes in Sierra Leone, Bignami and his colleagues, following the pioneering work of Grassi, had succeeded in infecting a healthy volunteer with P. falciparum from mosquito bites in Rome. Both Laveran and Ross received Nobel Prizes for their respective discoveries.

Understanding of the biology of malaria was further advanced by a third Nobel-prize winning discovery. In 1883 the Viennese psychiatrist Julius Wagner–Jarregg became interested in the relationship between fever and mental illness. Between 1888 and 1917 he experimented on a number of methods of inducing fever to treat patients with ‘general paralysis of the insane’ (GPI is a form of neurosyphilis). On 14 June 1917 he inoculated blood from a soldier with tertian fever into two patients with GPI. So began the era of malariatherapy of

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

Exoerythrocytic (hepatic) phase of development (days)

5.5 8 9 15 ?7

Erythrocytic cycle (days) 2 2 2 3 1Hypnozoites (relapses) No Yes Yes No NoNumber of merozoites per hepatic schizont 30 000 10 000 15 000 2000Erythrocyte preference Young RBCs but can

invade all agesaReticulocytes Reticulocytes Old RBCs All ages

Maximum duration of untreated infection (years) 2 4 4 40 ?

aParasites causing severe malaria are not selective in red cell invasion.

TABLE 43.1 Human Malaria Parasites

534 SECTION 9 Protozoan Infections

appear similar under light microscopy. The young developing parasite looks like a signet ring or, in the case of P. falciparum, like a pair of stereo-headphones, with darkly staining chroma-tin in the nucleus, a circular rim of cytoplasm and a pale central food vacuole. Parasites are freely motile within the erythrocyte. As they grow they increase in size logarithmically and consume the erythrocyte’s contents (most of which is haemoglobin). With this increase in size the P. falciparum parasitized erythro-cyte becomes spherical and less deformable. Proteolysis of hae-moglobin within the digestive vacuole releases amino acids which are taken up and utilized by the growing parasite for protein synthesis, but the liberated haem poses a toxic threat. Freed from its protein scaffold haem is highly reactive and readily oxidizes to the ferric form. Toxicity is avoided by spon-taneous and protein-facilitated dimerization to an inert crystal-line substance, haemozoin. Non-polymerized haem exits the food vacuole but is then degraded in the cytosol by glutathione. Excess non-polymerized haem overwhelms the defence mecha-nism and is toxic. The digested products, mainly the brown or black insoluble pigment haemozoin, can be readily seen within the digestive vacuole of the growing parasite. To obtain amino acids and other nutrients and to control the electrolytic milieu in the infected erythrocyte the parasite inserts specific trans-porters and other proteins in the red cell membrane. These and other disruptions make the red cell more permeable. The P. falciparum infected erythrocyte becomes progressively less elastic and deformable and more spherical as the parasite grows.

At approximately 12–14 hours of development P. falciparum parasites begin to exhibit a high-molecular-weight strain-specific variant antigen, Plasmodium falciparum erythrocyte membrane protein 1 (Pf EMP1) on the exterior surface of the infected red cell which mediates attachment to vascular endo-thelium. This is associated with knob-like projections from the erythrocyte membrane. Expression increases towards the middle of the cycle (24 hours). These ‘knobby’ or K+ red cells then progressively disappear from the circulation by attachment or ‘cytoadherence’ to the walls of venules and capillaries in the vital organs. This process is called ‘sequestration’. The other three ‘benign’ human malarias do not cytoadhere in systemic blood vessels and all stages of development circulate in the blood-stream.

As P. vivax grows it enlarges the infected red cell, which in contrast to P. falciparum, leads to an increase in deformability as the parasite matures. Red granules known as Schuffner’s dots appear throughout the erythrocyte. Similar dots are also promi-nent in P. ovale, which also distorts the shape of the infected erythrocyte (hence its name). P. malariae produces characteris-tic ‘band forms’ as the parasite grows. It is usually present at low parasitaemias. When humans are infected with the potentially lethal monkey malaria P. knowlesi it is often mistaken for P. malariae under light microscopy.1 High parasitaemias (over 2%) are usually caused by P. falciparum or P. knowlesi. Approxi-mately 36 hours after merozoite invasion (or 54 hours in P. malariae) repeated nuclear division takes place to form a ‘segmenter’ or schizont (the term ‘meront’ is etymologically more correct). Eventually the growing parasite occupies the entire red cell which has become spherical, depleted in haemo-globin and full of merozoites. This then ruptures; so that between 6 and 36 merozoites are released, destroying the rem-nants of the red cell. Following P. falciparum schizogony the residual cytoadherent erythrocyte membrane and associated malaria pigment often remains attached to the vascular

of development considerable asexual multiplication takes place and many thousands of merozoites are released from each rup-tured infected hepatocyte. However, as only a few liver cells are infected, this phase is asymptomatic for the human host.

ASEXUAL BLOOD-STAGE DEVELOPMENT

The merozoites liberated into the bloodstream closely resemble sporozoites. They are motile ovoid forms which invade red cells rapidly. The process of invasion involves attachment to the erythrocyte surface, orientation so that the apical complex (which protrudes slightly from one end of the merozoite and contains the rhoptries, the micronemes and dense granules) abuts the red cell and then interiorization takes place by a wrig-gling or boring motion inside a vacuole composed of the invagi-nated erythrocyte membrane. Once inside the erythrocyte the parasite lies within the erythrocyte cytosol enveloped by its own plasma membrane and a surrounding parasitophorous vacuo-lar membrane. The attachment of the merozoite to the red cell is mediated by attachment of one or more of a family of eryth-rocyte binding proteins, localized to the micronemes of the merozoite apical complex, to a specific erythrocyte receptor. In P. vivax and P. knowlesi this is related to the Duffy blood group antigen Fya or Fyb. The absence of these phenotypes in West Africans, or people who originate from that region, has been suggested to explain their proven resistance to infection with P. vivax and the absence of vivax malaria in West Africa. But early malariatherapy observations and recent epidemiological studies in continental Africa and Madagascar show that P. vivax can infect Duffy-negative individuals, so there are probably multi-ple invasion pathways. For P. falciparum the reticulocyte-binding protein homologue 5 (PfRh5) is indispensable for erythrocyte invasion. Basigin (CD147, EMMPRIN) has been identified as the erythrocyte receptor of PfRh5 and shown to be essential for the invasion of multiple strains.4 The merozoite protein EBA 175, a member of the ‘Duffy binding like’ (DBL) superfamily of genes encoding ligands for host cell receptors is also clearly important. This binds to sialic acid and the peptide backbone of the red cell membrane sialoglycoprotein glycopho-rin A. Other sialic-acid-dependent and -independent pathways of invasion also occur indicating considerable reserve in the invasion system. Binding is linked to activation of a parasite actin motor which provides the mechanical energy for the inva-sion process. The red cell surface receptors for P. malariae and P. ovale are not known.

During the early stage of intraerythrocytic development (<12 hours) the small ‘ring forms’ of the four parasite species

Prepatent Period (Days)

Incubation Period (Days)

P. falciparum 11.0 ± 2.4 13.1 ± 2.8P. vivax 12.2 ± 2.3 13.4 ± 2.7P. malariae 32.7a 34.7a

P. ovale 12.0 14.1

Values are mean ± SD.aThese data are taken from artificially induced malaria data in Boyd

(1948); naturally acquired infections are considered to have an incubation period of between 13–28 days.

TABLE 43.2

Malaria Incubation Periods in Malaria Therapy and Volunteer Studies

43  Malaria 535

individual genes were cloned and sequenced on the long and winding (and as yet unfinished) road towards the development of a malaria vaccine and in the past few years the entire genomes of several hundred malaria parasites have been sequenced. P. falciparum has approximately 5300 genes in its 14 chromo-somes and extrachromosomal elements compared with the 31 000 of its natural host. Codon composition is extremely biased to adenine and thymine in P. falciparum but more evenly balanced in the other malaria parasite genomes. There appear to be some groupings of genes related to function. For example, the genes encoding the merozoite surface proteins are grouped. The many genes encoding the variant red cell surface antigens (var and rif families), which contribute to the antigenic diversity necessary for the parasite to elude the host immune system, are also located close to each other near the telomeres. The var gene product, the variant surface protein which medi-ates cytoadherence (PfEMP1) appears to be the main antigen determining the parasite population structure during chronic falciparum malaria infections. Variation in surface antigenicity results from the activation of different var genes. This switching occurs at different rates, some of which exceed 2% per asexual cycle. It has been suggested that the diversity of these immuno-dominant variant repeat sequences interferes with the selection of high-affinity antibody responses and perpetuates low-affinity responses in malaria. This ‘confusion of the immune response’ delays the development of effective immunity. Immune selec-tion also provides the selective pressure to maintain diversity in T- and B-cell epitopes through a high frequency of non-synonymous base mutations during the asexual development of malaria parasites. At a larger scale, genetic changes resulting in drug resistance have had a profound effect on the malaria para-site population structure with the progeny of drug-resistant parasites originating in South-east Asia sweeping across India and then spreading across Africa.6

The mechanisms maintaining genetic diversity within the parasite genome are many and complex. Some of the polymor-phic antigens identified are encoded by single gene copies in the haploid genome. These polypeptide antigens are characterized by tandem repeat sequences. Unequal crossing over during recombination can generate completely different sequences of these repeats. As these repeat sequences are also antibody targets, their variation provides further antigenic diversity.

EpidemiologyMalaria infects approximately 5% of the world’s population and causes over six hundred thousand deaths each year. Most of these deaths are in African children. Malaria (both P. falciparum and P. vivax) also contributes to neonatal mortality by reducing birth weight.

DISTRIBUTION

Malaria is found throughout the tropics (Figure 43.1). In Africa, P. falciparum predominates, as it does on the island of New Guinea and in Haiti, whereas P. vivax is more common in Central and parts of South America, North Africa, the Middle East and the Indian subcontinent. The prevalence of both species is approximately equal in other parts of South America, South-east Asia and Oceania. P. vivax is rare in West Africa (but is common in the horn of Africa, whereas P. ovale is common only in West Africa. P. malariae is found in most areas, but is

endothelium for many hours. The released merozoites rapidly reinvade other red cells and start a new asexual cycle. Thus the infection expands logarithmically at approximately 10-fold per cycle. Only a sub-population of erythrocytes can be invaded, determined largely by red cell age. P. vivax can invade red cells for up to 2 weeks after emergence from the bone marrow. In Thailand, P. falciparum parasites causing severe malaria showed unselective invasion and had a greater multiplication potential at high densities than those which caused uncomplicated malaria. The asexual life cycle is approximately 24 hours for P. knowlesi, 48 hours for P. falciparum, P. vivax and P. ovale and 72 hours for P. malariae.

SEXUAL STAGES AND DEVELOPMENT IN THE MOSQUITO

After a series of asexual cycles in Plasmodium falciparum, a subpopulation of parasites develops into sexual forms (game-tocytes) which are long-lived and motile. These are the stages which transmit the infection. The process of gametocytogony takes about 7–10 days in P. falciparum but only 4 days in P. vivax which begins gametocytogenesis immediately in the blood stage infection. Thus there is an interval of approximately 1 week between peak asexual and sexual stage parasitaemia in acute falciparum malaria. There is no delay with P. vivax so symptom-atic P. vivax infections are more likely to present with patent gametocytaemia before treatment (and therefore to transmit) than acute P. falciparum infections. The male-to-female game-tocyte sex ratio for P. falciparum is approximately 1 : 4, although each male gametocyte can produce up to 8 microgametes each capable of individual fertilization. Following ingestion in the blood meal of a biting female anopheline mosquito, the male and female gametocytes become activated in the mosquito’s gut.5 The male gametocytes undergo rapid nuclear division and each of the eight nuclei formed associates with a flagellum (20–25 mm long). The motile male microgametes then separate and seek the female macrogametes. Fusion and meiosis then take place to form a zygote. For a brief period the malaria para-site is diploid. Within 24 hours the enlarging zygote becomes motile and this form (the ookinete) penetrates the wall of the mosquito mid-gut (stomach) where it encysts (as an oocyst). This spherical bag of parasites expands by asexual division to reach a diameter of approximately 500 µm, i.e. it becomes visible to the naked eye. During the early stage of oocyst devel-opment there is a characteristic pigment pattern and colour that allows speciation (it was this that caught the eye of its discov-erer, Ronald Ross, in 1894), but these patterns become obscured by the time the oocyst has matured to contain thousands of fusiform motile sporozoites. The oocyst finally bursts to liberate myriads of sporozoites into the coelomic cavity of the mos-quito. The sporozoites then migrate to the salivary glands to await inoculation into the next human host during feeding. The development of the parasite in the mosquito is termed spo-rogony and takes between 8 and 35 days depending on the ambient temperature and species of parasite and mosquito. The longevity of the mosquito is a critical factor in determining its vectorial capacity (see above).

MOLECULAR GENETICS

Inheritance in Plasmodium is similar to that in other eukaryotes. Haploid and diploid generations alternate. A large number of

536 SECTION 9 Protozoan Infections

America, Europe, North Africa, the Middle East, parts of North India, central China and North Korea P. vivax has (or had) a different relapse periodicity; after the initial illness the first relapse is 8–10 months later. In Northern Europe and Northern Russia the interval between inoculation and first illness was 8–10 months (P. vivax hibernans).3

relatively uncommon outside Africa. Malaria was once endemic in Europe and northern Asia and was introduced to North America, but it has been eradicated from these areas. In South and South-east Asia, Oceania and South America P. vivax relapses at frequent regular intervals (of 3 weeks if rapidly elim-inated antimalarial drugs are given). In North and Central

Figure 43.1 Global distribution of Plasmodium falciparum and Plasmodium vivax malaria. Upper panel shows the model-based geostatistical point (MBG) estimates of the Plasmodium falciparum annual mean parasite rate PfPR2–10 (defined as the predicted proportion of 2–10-year-olds with patent parasitaemia) for 2010 within the spatial limits of stable P. falciparum malaria transmission.40 Lower panel shows equivalent estimates of the Plasmodium vivax annual mean parasite rate (PvPR1–99).41 Note this prediction is for all age groups (1–99). P. vivax is rare in areas with a high preva-lence of Duffy (blood group) negativity.

>70%

0%Unstable transmission Risk free

PfPR 2-10

>7%

0%Unstable transmissionUnstable transmissionand high Duffy negativity Risk free

PvPR 1-99

43  Malaria 537

the tenth power of the probability of the mosquito surviving for 1 day. The model described by MacDonald has certain theo-retical limitations (it has been refined in recent years to accom-modate these), but it does illustrate certain fundamental points of practical relevance to control or eradication programmes. Vector longevity in determining transmission is clearly of central importance and focuses control measures on the adult mosquito. At very high levels of transmission there is consider-able reserve in the system and large reductions in transmission reduce malaria by a negligible amount (e.g. a reduction in transmission of 90% from 300 infectious bites per year to 30 bites/year will make very little difference to the prevalence of malaria) – but as r0 approaches the critical value of 1 (below which the disease dies out), small reductions in r0 have very large effects on the amount of malaria. Thus, malaria is poten-tially very vulnerable in low-transmission settings. Control pro-grammes can be very effective in these circumstances and can eradicate malaria – as indeed they did in Europe where r0 was certainly low in many areas, drug treatment was freely available, and the vector rested inside houses and could be attacked with residual insecticides. Vectors differ considerably in their natural abundance (particularly with season of the year), feeding and resting behaviours, breeding sites, flight ranges, choice of blood source (many anopheline vectors also bite animals), and vulner-ability to environmental conditions and insecticides.

There is also considerable variation in the ability of different anopheline mosquito species to transmit malaria (the vectorial capacity). There are nearly 400 species of anopheline mosqui-toes and many are species complexes. Confusingly, the taxon-omy continually changes as differences within species complexes are characterized and molecular genetics reveals their phylog-eny. Approximately 80 anopheline species can transmit malaria, 66 are considered natural vectors, and about 45 are considered important vectors. Each vector has its own behaviour patterns, and even within a species these can vary between geographic areas and can change with selection pressures (such as insecti-cide use). For example in South-east Asia mosquitoes of the Anopheles dirus complex are an important cause of ‘forest and forest fringe’ malaria. They breed in the tree collections of water and are consequently vulnerable to deforestation, or too little or too much rainfall, but they are very difficult to attack with insecticides. A. sundaicus and A. epiroticus are found near the coast as they breed in brackish water. Human biting times vary considerably within the species complexes. A. stephensi, the principal vector in the Indian subcontinent, breeds in wells or stagnant water and can be controlled by treating breeding sites with insecticides or polystyrene balls. The most effective malaria vectors (such as the A. gambiae complex in Africa) are hardy, long-lived, naturally occur in high densities and bite humans frequently. Malaria is often seasonal, coinciding with the rainy season which provides water for mosquito breeding and increased humidity favouring mosquito survival. Other factors, which are not well understood, also influence mosquito popula-tions and lead to fluctuations in the prevalence of malaria.

THE HUMAN HOST

The behaviour of man also plays an important role in the epi-demiology of malaria. There must be a human reservoir of viable gametocytes to transmit the infection. In areas of high transmission infants and young children are more susceptible to malaria than the more immune older children and adults.

THE MOSQUITO VECTOR

Malaria is transmitted by some species of anopheline mosqui-toes. Malaria transmission does not occur at temperatures below 16°C, or above 33°C, and at altitudes >2000 m because development in the mosquito (sporogony) cannot take place. The optimum conditions for transmission are high humidity and an ambient temperature between 20°C and 30°C. Although rainfall provides breeding sites for mosquitoes, excessive rainfall may wash away mosquito larvae and pupae.

The epidemiology of malaria is complex and may vary con-siderably even within relatively small geographic areas. In low-transmission settings small foci of much higher transmission sustain malaria and confound elimination efforts. Malaria transmission to man depends on several interrelated factors. The most important pertain to the anopheline mosquito vector and in particular its longevity. As sporogony (development of the sporozoite parasites in the vector) takes over a week (depend-ing on ambient temperatures), the mosquito must survive for longer than this after feeding on a gametocyte-carrying human, if malaria is to be transmitted. Macdonald gave the following formula for the likelihood of infection based on the sporozoite rates, i.e. the proportion of anopheline mosquitoes with sporo-zoites in their salivary glands.

S =−P ax

ax P

n

elog

where P = the probability of mosquito survival through 1 day; n = the duration, in days, of the extrinsic cycle of the parasite in the mosquito; a = average number of blood meals on man per day and x = the proportion of bites infective to man. The probability of a mosquito surviving n days is given by:

P

P

n

e− log

The inoculation rate, or the mean daily number of bites (h) received by sporozoite-bearing mosquitoes is given by:

h mabs=

where m = anopheline density in relation to man and b = pro-portion of bites that are infectious. The reproductive rate of the infection (r) or the number of secondary cases resulting from a primary case is then given by:

rma bP

z P

ax

ax P

n

e e

=−

−−

2

1log log

where z is the recovery rate, or the reciprocal of the duration of human infectivity. This is usually estimated at 80 days for P. falciparum in a non-immune subject, i.e. z = 0.0125. The term:

1−−

ax

ax Pelog

refers to the proportion of anopheline mosquitoes ‘not yet infected’. When transmission is very low (i.e. x approaches 0) then the basic reproductive rate (r0) reduces to:

rma bP

z P

n

e0

2

=− log

Thus, as a general approximation, malaria transmission is directly proportional to the density of the vector, the square of the number of times each day that the mosquito bites man and

538 SECTION 9 Protozoan Infections

erythrocytes which retards parasite development. In holoen-demic areas the baby is inoculated repeatedly with sporozoites during the first 6 months of life, but the blood stage infection is seldom severe. People may receive up to three infectious bites per day. In this epidemiological context the main clinical impact of falciparum malaria is to cause severe anaemia in the 1–3-year age group (Figure 43.2). With less intense or more variable or unstable transmission the age range affected by severe malaria extends to older children and cerebral malaria becomes a more prominent manifestation of severe disease.9 Although mortality falls with decreasing transmission intensity, it remains substan-tial until the EIR falls well below one. In hyperendemic and holoendemic areas indigenous adults never develop severe malaria, unless they leave the transmission area and return years later (and even then malaria is seldom life-threatening). Immu-nity is constantly boosted and effective premunition prevents parasite burdens reaching dangerous levels. Nearly all infections in adults are asymptomatic. In terms of the mathematical models presented earlier, an index of malaria transmission sta-bility is given by al-logeP; values of >2.5 indicate stable transmission.

Where transmission of malaria is low, erratic, markedly sea-sonal, or focal, symptomatic infections are more common. A state of premunition is often not attained. Symptomatic disease occurs at any age and cerebral malaria is a prominent manifes-tation of severe disease at all ages. This is termed ‘unstable’ malaria. Epidemics with high mortality may occur. In many areas the transmission of malaria varies considerably over short distances and severe disease is common when non-immune individuals enter these areas (e.g. woodcutters in South America and South-east Asia where malaria is of the ‘forest fringe’ type, or highland refugees in Burundi descending into malarious lowlands).With declining malaria as malaria control efforts are successful the clinical epidemiology of malaria changes and older children and adults are increasingly likely to develop symptomatic illness.

Malaria is usually a ‘rainy season disease’ coinciding with increased mosquito abundance. In some areas parasite rates (i.e. the proportion of people with positive blood smears) are rela-tively constant throughout the year, but the majority of cases

Parasite densities are higher and gametocytaemia is detected more frequently in children. In endemic areas the relative con-tributions to overall transmission of the younger age group, who have higher parasite densities, become ill more often and are therefore more likely to receive drugs, versus the older asymptomatic individuals who have lower parasite densities and also immunity and are less likely to receive antimalarial treatment, is unclear. The endemicity of malaria is best defined by the entomological inoculation rate (EIR), or number of infectious mosquito bites received per person per year (although this is difficult to measure accurately), but is defined tradition-ally in terms of the spleen or parasite rates in children aged between 2 and 9 years.

• Hypoendemic: spleen rate or parasite rate 0–10%• Mesoendemic: spleen or parasite rate 10–50%• Hyperendemic: spleen or parasite rate 50–75% and adult

spleen rate is also high• Holoendemic: spleen or parasite rate over 75% and

adult spleen rate low. Parasite rates in the 1st year of life are high.

In areas which are holoendemic or hyperendemic for P. falci-parum, such as much of tropical Africa or coastal New Guinea, people are infected repeatedly throughout their lives. There is considerable morbidity and mortality during childhood. In The Gambia, where people were infected once each year on average (a relatively low figure for the African continent), malaria was estimated to cause 25% of deaths between 1 and 4 years of age. The effects of insecticide-treated nets on death rates in children (average reduction in all-cause mortality in children under 5 years old of approximately 20%) across sub-Saharan Africa is further testament to the impact of malaria on child survival. But eventually, if the child survives, a state of ‘premunition’ is achieved where infections cause little or no problems to the host. Thus a form of immunity develops which is sufficient to control, but not prevent, the infection. The slow rate at which premunition is acquired may be a function of age. Non-immune adults entering an area of intense transmission acquire premu-nition more rapidly than children. Falciparum malaria infec-tions are more severe in pregnancy, particularly in primigravidae, and appears to be augmented by iron supplementation.

It is difficult to be precise about how many people die each year from malaria, as the disease is most prevalent where health services are lacking. But in recent years a considerable effort has gone into deriving estimates of the global burden of disease.7 It is widely quoted that 90% of the deaths from malaria in the world are in African children, but recent studies suggest that the burden of malaria in Asia may have been under-estimated.8 It has also been claimed that there is a large and previously unrecognized mortality from malaria in older adults, but this is misleading and reflects the lack of specificity of the verbal autopsy approach to ascertaining cause of death. The setting up of standardized demographic surveillance systems in many malaria-endemic areas has resulted in more accurate data and more accurate measurement of the impact of interventions.

CLINICAL EPIDEMIOLOGY

Babies develop severe malaria relatively infrequently (although, if they do, the mortality is high). The factors responsible for this apparent protection include passive transfer of maternal immunity and the high haemoglobin F content of the infants’

Figure 43.2 Relationship between age and the clinical presentations of severe falciparum malaria at different levels of malaria transmission.

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43  Malaria 539

invasion by malaria parasites and provide a hostile intraeryth-rocytic ionic milieu for development. Haemoglobin E hetero-zygotes (HbAE) are haematologically almost normal and these individuals are susceptible to falciparum malaria but appear to be protected against severe malaria. Parasite multiplication at high densities is reduced. G6PD deficiency reduces parasite densities in P. vivax infections.12

Apart from the well-established protection conferred by polymorphisms in the genes encoding haemoglobin, a large and confusing array of other polymorphisms associated with pro-tection and susceptibility to malaria have been reported. In some cases a polymorphism has been associated with protec-tion in one study and susceptibility in another! Three different TNF promoter polymorphisms appear independently to be associated with severe malaria; Gambian children homozygous for the TNF-308A allele were at a sevenfold increased risk of dying or recovering with neurological sequelae. Although this association was confirmed in East Africa it was not found in two independent studies in Asia. TNF-238A was associated independently with severe anaemia and TNF-376A with suscep-tibility to cerebral malaria. A single nucleotide polymorphism in the inducible nitric oxide synthase gene promoter region was associated with severe anaemia in Gabon. Separate case–control studies on genetic polymorphisms in CD36 and ICAM-1, the two major receptors for P. falciparum cytoadherence have given conflicting results. The CD36 polymorphism protected from severe malaria in one study but increased the risk in the other. An African ICAM-1 polymorphism predisposed to cerebral malaria in Kenya, was neutral in The Gambia, and protected in Gabon. In some of these associations the possibility of linkage cannot be ruled out (i.e. the polymorphic gene lies close to another gene which is causally associated with the observed effect). For example the MHC III region, where the TNF pro-moter polymorphisms are located, contains a remarkably high density of genes with probable immune functions. The contri-bution of epistasis, which is the interaction between genes, to malaria susceptibility and resistance has been underappreci-ated. This makes interpretation of genetic associations very dif-ficult and probably explains many of the inconsistencies described above.

PathologyEXPANSION OF THE INFECTION

When the hepatic schizonts rupture, they liberate approxi-mately 105–106 merozoites into the circulation (i.e. the product of 5–100 successful sporozoites). These invade passing red cells immediately. In non-immune subjects the multiplication rate in P. falciparum usually approximates 10 per cycle (i.e. >50% efficiency) but may reach twenty-fold per cycle during the expanding phase of the infection (Figure 43.3). For the first few cycles the host is unaware of the brewing infection. On average, parasites are detectable in the blood by microscopy on the 11th day after sporozoite inoculation (the diligent micros-copist can detect 20–50 parasites/µL reliably on Giemsa-stained thick films). At this stage the host may still feel well, or may complain of vague non-specific symptoms of malaise, head-ache, myalgia, weakness or anorexia. On average the fever begins 1–2 days later, but in some cases fever precedes detect-able parasitaemia. The rise in parasite count is logarithmic ini-tially, with a rising sine wave pattern of parasitaemia in

still do occur during the wet season. Deforestation, population migration and changes in agricultural practice have profound effects on malaria transmission. Urban malaria is becoming an increasing problem in many countries. Malaria can also behave as an epidemic disease carrying a high mortality. Epidemics are caused by migrations (i.e. introduction of susceptible hosts), the introduction of new vectors, or changes in the habits of the mosquito vector or the human host. Epidemics have occurred in North India, Sri Lanka, South-east Asia, Ethiopia, Madagas-car, Brazil (when the formidable African vector A. gambiae was inadvertently imported from Africa in the 1930s) and more recently in Burundi and KwaZulu Natal, where drug resistance was also a contributory factor.

Increasing international air travel and worsening antima-larial drug resistance have led to an increase in imported cases of malaria in tourists, travellers, and immigrants. With the recent exception of some of the former Soviet republics in East Europe and West Asia, this has not led to the reintroduction of malaria to areas from where it had earlier been eradicated (although the vector, and thus the potential, remains). Imported malaria is often misdiagnosed, leading to delays in treatment and severe presentations of falciparum malaria are not uncom-mon. Malaria may also be transmitted by blood transfusion, transplantation, or through needle-sharing among intravenous drug addicts.

GENETIC FACTORS PROTECTING AGAINST MALARIA

In 1949, JBS Haldane suggested that people who were hetero-zygous for red cell abnormalities such as thalassaemia or sickle cell disease might be protected against malaria. This, he said, would explain the high gene frequencies for the haemoglobin-opathies in tropical areas and their rarity in colder climates. A state of ‘balanced polymorphism’ would exist, whereby the loss of the disadvantaged homozygotes would be offset by the sur-vival advantage in heterozygotes. There is now good evidence from detailed epidemiological studies that this hypothesis is correct. The greatest protection is conferred by sickle cell trait and Melanesian ovalocytosis. These patients’ cells resist parasite invasion (in the case of sickle cell trait under low oxygen ten-sions) and once invaded the AS cells sickle readily, facilitating their clearance by the reticuloendothelial system. P. falciparum-infected red cells containing haemoglobins S and C show reduced cytoadherence because of reduced presentation of the ligand Pf EMP1. The protective effect conferred by the thal-assaemias or glucose-6-phosphate dehydrogenase (G6PD) defi-ciency (which share a geographical distribution with malaria) is generally weaker. The main protection is against severe malaria (Hb AS, CC, AC, AE, homozygous and heterozygous alpha thalassaemia, G6PD deficiency).10

The mechanism of protection in many of these haemoglo-binopathies, and how they interact with each other, is still incompletely understood.11 The rate of decline of haemoglobin F in the 1st year of life is slower in α- and β-thalassaemia het-erozygotes. Erythrocytes containing high haemoglobin F con-centrations do not support parasite growth well. But studies from Vanuatu indicate that children with α-thalassaemia actu-ally have more malaria (both P. falciparum and P. vivax) in the early years of life than their ‘normal’ counterparts suggesting a complex interaction between malaria species and haemoglobin chain synthesis. Melanesian ovalocytic erythrocytes both resist

540 SECTION 9 Protozoan Infections

descriptions of malaria symptomatology derive largely from detailed clinical observations made in the late nineteenth and early twentieth centuries, the experience with artificial infec-tions in early chemotherapy trials, studies conducted by the military, and the extensive use of malaria therapy in the treat-ment of neurosyphilis. These observations were usually made on non-immune adults. In malaria therapy patients with neu-rosyphilis were artificially infected, by mosquito bite or transfu-sion and the infection with P. falciparum or P. vivax was left untreated so that the patient experienced recurrent high fevers, or if symptoms were severe, was judiciously titrated with quinine. Nowadays, these characteristic fever charts with regular fever spikes are seen rarely because malaria is treated promptly. It was also apparent from these studies and later animal experi-ments that some strains of P. falciparum and P. vivax were more virulent than others. For example, the now extinct European strains of P. falciparum were notorious. The virulence factors of malaria parasites have not been characterized fully, but include multiplication capacity, cytoadherence and rosetting ability, the potential to induce cytokine release, antigenicity, and antima-larial drug resistance.

PARASITE BIOMASS

Malaria is readily diagnosed from the blood film stained with a Romanowsky dye. In the benign malarias (where sequestration is considered not to occur) the number of parasites in the body may be estimated simply by multiplying the parasitaemia by the estimated blood volume. In P. falciparum infections the micros-copist can see only the first third of the asexual life cycle. In the second two thirds the parasitized cells are sequestered in the capillaries and venules. As a consequence, there may be large discrepancies between the number of parasites in the peripheral (circulating) blood and the number of parasites in the body (the parasite burden) (Figure 43.4). This has often puzzled and mis-led clinicians; some patients appear to tolerate high para-sitaemia with little adverse effects, whereas others die with low

falciparum malaria, but in most cases the parasite expansion terminates abruptly to limit the infection at a parasitaemia of 104–105/uL (Figure 43.3). Only P. falciparum and P. knowlesi have the capacity for untrammelled multiplication and parasi-taemias may exceed 50% in some cases. Several factors converge to limit parasite multiplication. The host mobilizes specific and nonspecific immune defences (particularly in the spleen). The parasite schizonts are also damaged by high fevers. The avail-ability of suitable red cells is exhausted: P. vivax and P. falci-parum prefer younger red cells and P. malariae prefers older cells. Interestingly whereas P. vivax shows invasion restricted to only 13% of the red cell population and P. falciparum causing uncomplicated malaria to 40%, P. falciparum parasites causing severe malaria in South-east Asia show unrestricted invasion. Thus the untreated infection increases exponentially, then the rate of expansion decelerates rapidly, parasitaemia fluctuates, settles around a plateau, then declines and continues for several weeks to several years at low levels before finally being elimi-nated. Although natural infections often contain two or more genetically different parasite strains, development tends to be relatively synchronous from the outset. Further synchroniza-tion takes place in untreated infections in non-immune sub-jects, such that merogony (‘sporulation’) takes place within 1–2 hours. This is associated with fever and rigors (the ‘parox-ysm’). Although one ‘brood’ predominates, in P. falciparum there is often at least one minor ‘brood’ or subpopulation cycling 24 hours out of phase with the major brood.

The periodicity of malaria is enshrined in the terminology of the fever pattern. P. malariae has a 72-hour life cycle and in untreated infections the paroxysm occurred on the 4th day (using the Greek system of ‘inclusive reckoning’ the previous paroxysm is considered to occur on day 1). This is termed ‘quartan malaria’. The other malarias are termed tertian (fever on the 3rd day; 48-hour asexual cycle). P. falciparum often syn-chronized to a daily fever spike (quotidian fever), presumably caused by two broods of approximately equal size oscillating 24 hours out of phase, or failed to synchronize at all. The classic

Figure 43.3 Logarithmic expansion of malaria infections in vivo. The body burden represents the total number of parasites in the body fol-lowing infection of an adult of 50 kg. In falciparum malaria the infection rapidly reaches a lethal burden at high multiplication rates unless restrained. Maximum recorded multiplication rates are approximately ×20/cycle in vivo.

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Figure 43.4 The problem of assessing the parasite burden from the peripheral parasitaemia in P. falciparum malaria. Sequestration hides the parasites causing harm. Two patients (A and B) have the same para-sitaemia. In patient A, most of the parasites are circulating and only a few from the previous cycle have yet to undergo merogony (schizont rupture). In patient B, most of the parasites have already sequestered and only 20% of the biomass still circulates. There are over 60 times more parasites in patient B than in patient A. The clue lies in the stage distribution (shown crossing the hatched lines) of the circulating para-sites which will be more mature in patient B.

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43  Malaria 541

THE IMMUNE RESPONSE

Following natural infection there is a transient humoral response to sporozoite antigens; sporozoite antibodies decline, with a half-life of 3–4 weeks. In areas of high transmission sporozoite antibody levels tend to plateau between 20 and 30 years of age and do not correlate with premunition. Cytotoxic T-cell immune responses cannot be directed against the blood-stage parasite as red cells do not express human leukocyte (HLA) antigens, but the pre-erythrocytic liver stages of the parasite are vulnerable to T cell attack. Several lines of experi-mental evidence in animal malarias and the observation that certain HLA types are relatively protected from severe malaria, indicate that class 1 restricted CD8(+) T-cells play an important role in immunity. There is evidence supporting a role for both alpha-beta and gamma-delta CD4+ cells in the immune response to malaria.

Strain-specific immunity to the asexual blood-stage para-sites develops slowly during natural untreated infections, but it then provides good protection against rechallenge. However, parasite populations are diverse and cross-strain protection is initially weak or negligible. The development of immunity in endemic areas may represent the gradual acquisition of a rep-ertoire of immunological memory for the range of local para-sites. This involves strain transcending immunity sufficient to ameliorate disease (antitoxic immunity) and a more strain-specific immunity which protects from or attenuates the infection. The immune response to malaria is clearly very complex and the relative importance of humoral and cellular immunity in man has not been defined clearly. Infusion of hyperimmune serum to patients with acute malaria can reduce or eliminate parasitaemia mainly through opsonization and activation of phagocytic and cytotoxic effector functions by cytophilic IgG antibodies and augmentation of ring-form infected erythrocyte clearance. Immune serum also reduces parasite multiplication by agglutinating merozoites. In addition to the role of cellular immunity in preventing preerythrocytic development, the relatively weak but definite increase in malaria severity in patients living in endemic areas with the acquired immune deficiency syndrome (HIV-AIDS) suggests that CD4+ cells do play a significant role in modulating the severity of falciparum malaria.

PathophysiologyThe pathophysiology of malaria results from destruction of erythrocytes, the liberation of parasite and erythrocyte material into the circulation, and the host reaction to these events. P. falciparum malaria-infected erythrocytes sequester in the microcirculation of vital organs, interfering with microcircula-tory flow and host tissue metabolism.

TOXICITY AND CYTOKINES

For many years malariologists hypothesized that parasites con-tained a toxin which was liberated at schizont rupture and caused the symptoms of the paroxysm. No toxin in the strict sense of the word has ever been identified, but malaria parasites do induce release of cytokines in much the same way as bacte-rial endotoxin. A glycolipid material with many of the proper-ties of bacterial endotoxin is released on meront rupture. This material is associated with the glycosylphosphatidylinositol

parasite counts. The clue to the discrepancy lies both in the immune status of the host and in the stage of development of parasites on the peripheral blood smear. A predominance of more mature parasites indicates that a greater proportion is sequestered and carries a worse prognosis for any parasitaemia than a predominance of younger forms. Two patients with the same peripheral parasitaemia may have as much as a one- hundred-fold difference in the total number of parasites in the body. The presence of intraneutrophilic phagocytosed malaria pigment (in more than 5% of neutrophils) also reflects the degree of previous schizogony and is also a valuable prognostic index. Measurement of proteins released by the parasite such as Pf HRP2 provide a good method of assessing this hidden patho-genic sequestered biomass.13 In synchronous P. falciparum infections the peripheral blood parasite numbers fall at the time of sequestration and rise abruptly at the time of merogony (when a predominance of tiny rings are seen). The other expla-nation for the ability to tolerate high parasitaemias without apparent adverse effects relates to the development of ‘anti-toxic’ immunity. The host adapts to repeated infection by pro-ducing less cytokines for a given quantum of parasites (see below). Eventually a state is reached where infections are asymp-tomatic. This is called premunition.

IMMUNITY

The precise mechanisms controlling malaria infections are still incompletely understood. It was apparent from the era of malaria therapy for neurosyphilis, that a strain-specific immu-nity developed which protected against rechallenge with the same parasite strain, but did not protect from challenge with a different strain. Effective immunity, as distinct from premuni-tion, may be reached when there has been exposure to all local strains of malaria parasites. This is difficult to quantify as there is still no good in vitro correlate of either antitoxic or strain-specific immunity to malaria. In controlling the acute infection nonspecific host defence mechanisms and the later develop-ment of more specific cell-mediated and humoral responses are both important. Protective antibodies inhibit parasite expan-sion by agglutinating merozoites and through cooperation with the monocyte-macrophage series by binding to parasitized erythrocytes and then activating these cells’ Fc receptors. Non-specific effector mechanisms include non-opsonic phagocytosis via direct binding to monocyte-macrophage CD36, pro-inflammatory cytokine release, and the activation of phagocytic cells (including neutrophils) to release toxic oxygen species and nitric oxide, both of which are parasiticidal. The reaction of these oxygen intermediates with lipoproteins produces lipid peroxides. These are more stable cytotoxic molecules and are unaffected by antioxidants. There is also augmentation of splenic clearance function: both filtration and Fc receptor-mediated phagocytosis are increased. P. falciparum-infected erythrocytes are more rigid and more opsonized than unin-fected red cells as they express both host- and parasite-derived neoantigens on the erythrocyte surface. However, the parasite proteins expressed on the red cell surface undergo antigenic variation,14 and this is instrumental in avoiding complete immune clearance and sustaining the untreated infection. The systemic and splenic monocyte-macrophage series appear to be the most important immune effector cells in the direct attack on parasitized erythrocytes and merozoites, although neutro-phils may also play a role.

542 SECTION 9 Protozoan Infections

indicated that children with the (308A) TNF2 allele, a polymor-phism in the TNF promoter region, had a relative risk of 7 for death or neurological sequelae from cerebral malaria.15 This finding was not confirmed in studies from South-east Asia. A separate polymorphism in this region which affects gene expres-sion was associated with a fourfold increased risk of cerebral malaria. On the other hand the clinical studies in cerebral malaria with anti-TNF antibodies and other strategies to reduce TNF production, reported to date have shown no convincing effects other than reduction in fever. Cytokines do play a causal role in the pathogenesis of cerebral symptoms in murine models of severe malaria, but these models are clinically and pathologi-cally unlike human cerebral malaria. There is no direct evidence that systemic release of TNF or other cytokines causes coma in humans (although mechanisms involving local release of nitric oxide and other medicators within the central nervous system and consequent inhibition of neurotransmission can be hypoth-esized). In a large prospective study in adults with severe malaria, elevated plasma TNF concentrations were associated specifically with renal dysfunction and TNF levels were actually lower in patients with pure cerebral malaria than those with other manifestations of severe disease. Severe malarial anaemia has been associated with a different TNF promoter polymor-phism (238A; odds ratio 2.5). Taken together these suggest some role for TNF and other cytokines in severe disease, not encepha-lopathy per se, but the extent to which this is the cause or an effect of severe disease remains to be determined.

Cytokines are probably involved in placental dysfunction, suppression of erythropoiesis and inhibition of gluconeogene-sis, and they certainly do cause fever in malaria. Tolerance to malaria, or premunition, reflects both immune regulation of the infection and also reduced production of cytokines in response to malaria (‘antitoxic immunity’). Cytokines upregu-late the endothelial expression of vascular ligands for P. falciparum-infected erythrocytes, notably ICAM-1 and thus promote cytoadherence. They may also be important mediators of parasite killing by activating leukocytes and possibly other cells, to release toxic oxygen species, nitric oxide, and by gener-ating parasiticidal lipid peroxides and causing fever. Thus, whereas high concentrations of cytokines appear to be harmful, lower levels probably benefit the host.

SEQUESTRATION

Erythrocytes containing mature forms of P. falciparum adhere to microvascular endothelium (‘cytoadherence’) and thus dis-appear from the circulation. This process is known as sequestra-tion (Figure 43.5).14 The simian malaria parasites P. coatneyi and P. fragile infecting rhesus monkeys also sequester, but this does not occur to a significant extent with the other human malaria parasites. Sequestration is thought to be central to the pathophysiology of falciparum malaria. The mechanics of cytoadherence are similar to leukocyte endothelial interactions. Tethering (the initial contact) is followed by rolling and then firm adherence (stasis). Once adherent, the parasitized cell remains stuck until schizogony and even afterwards the residual membranes (and often the attached pigment body) remain attached to the vascular endothelium. Rolling is probably the rate-limiting factor determining cytoadherence.

Blood is a complex mixture of deformable cells suspended in plasma proteins, electrolytes, and a variety of small organic molecules. Its effective viscosity changes nonlinearly under the

anchor which covalently links proteins including the malaria parasite surface antigens to the cell membrane lipid bilayer. This activates host inflammatory responses in macrophages by sig-nalling through toll-like receptor (TLR) 2 and to a lesser extent TLR 4. Malaria-antigen-related IgE complexes also activate cytokine release. The limulus lysate assay, a test of endotoxin-like activity, is often positive in acute malaria. These products of malaria parasites and the crude malaria pigment which are released at schizont rupture, induce activation of the cytokine cascade in a similar manner to the endotoxin of bacteria. But they are considerably less potent. For example an E. coli bacter-aemia of 1 bacterium/mL carries an approximate mortality of 20% whereas in falciparum malaria only parasite densities of well over 109/mL produce such a lethal effect. Clearly compared with bacteria, malaria parasites are notable for their lack of toxicity! Cells of the macrophage-monocyte series, gamma/delta T cells, alpha/beta T cells, CD14+ cells and endothelium are stimulated to release cytokines in a mutually amplifying chain reaction. Initially tumour necrosis factor (TNF), which plays a pivotal role, interleukin (IL)-1 and gamma interferon are produced and these in turn induce a cascade of other ‘pro-inflammatory’ cytokines including IL-6, IL-8, IL-12, IL-18. These are balanced by production of the ‘anti-inflammatory’ cytokines notably IL-10 and related cytokines. Inflammatory cytokines are responsible for many of the symptoms and signs of the infection, particularly fever and malaise. Plasma concen-trations of cytokines are elevated in both acute vivax and falci-parum malaria. In established vivax malaria, which tends to synchronize earlier than P. falciparum, a pulse release of TNF occurs at the time of schizont rupture and this is followed by the characteristic symptoms and signs of the ‘paroxysm’, i.e. shivering, cool extremities, headache, chills, a spike of fever and sometimes rigors followed by sweating, vasodilatation and defervescence. For a given number of parasites Plasmodium vivax is a more potent inducer of TNF release than P. falci-parum, which may explain its lower pyrogenic density.

Whether pro-inflammatory cytokines contribute directly to the pathology of severe malaria remains uncertain. Cytokine concentrations in the blood fluctuate widely over a short period of time and are high in both P. vivax and P. falciparum; indeed some of the highest TNF concentrations recorded in malaria occur during the paroxysms of synchronous P. vivax infections. Nearly all the TNF measured in these assays is bound to soluble receptors; there is usually little or no bioactiv-ity. Nevertheless, in most series there is a positive correlation between cytokine levels and prognosis in severe falciparum malaria. Acute malaria is associated with high levels of most cytokines but the balance differs in relation to severity. IL-12 and TGF-β 1, which may regulate the balance between pro- and anti-inflammatory cytokines, are higher in uncomplicated than severe malaria. IL-12 is inversely correlated with plasma lactate – a measure of disease severity. IL-10, a potent anti-inflammatory cytokine, increases markedly in severe malaria but, in fatal cases, does not increase sufficiently to restrain the production of TNF. A reduced IL-10/TNF ratio has also been associated with childhood malarial anaemia in areas of high transmission. All this points to a disturbed balance of cytokine production in severe malaria.

The first studies to associate elevations in plasma cytokine levels with disease severity focussed on TNF and cerebral malaria and led to the suggestion that TNF played a causal role in coma and cerebral dysfunction. Genetic studies from Africa

43  Malaria 543

haematocrit rose from 10% to 20% and cytoadhesion rose 12-fold between 10% and 30%. Over this range, the viscosity of blood approximately doubles and so if shear stress is held con-stant, shear rates fall by approximately half allowing greater time for contact between cells and endothelium. The higher the haematocrit, the more cells roll along the endothelial surface and so a higher proportion of these adhere to the vascular endothelium.

Once infected red cells adhere, they do not enter the circula-tion again, remaining stuck until they rupture at merogony (schizogony). Under febrile conditions cytoadherence begins at approximately 12 hours after merozoite invasion and reaches 50% of maximum between 14–16 hours. Adherence is essen-tially complete in the second half of the parasites’ 48-hour asexual life cycle. As a consequence, whereas in the other malar-ias of man mature parasites are commonly seen on blood smears, these forms are rare in falciparum malaria and often indicate serious infection. It was thought that ring-stage-infected erythrocytes do not cytoadhere at all, but pathological and laboratory studies show that that they do, although much less so than more mature stages. Ring-form-infected parasites are also concentrated in the spleen and placenta, raising the intriguing possibility that the entire asexual cycle could take place away from the peripheral circulation. Sequestration occurs predominantly in the venules of vital organs. It is not distrib-uted uniformly throughout the body, being greatest in the brain, particularly the white matter, prominent in the heart, eyes, liver, kidneys, intestines and adipose tissue, and least in the skin. Even within the brain the distribution of sequestered erythrocytes varies markedly from vessel to vessel, possibly reflecting differences in the expression of endothelial receptors. Cytoadherence and the related phenomena of rosetting and autoagglutination lead to microcirculatory obstruction in falci-parum malaria (Figure 43.6). The gross microcirculatory obstruction caused by cytoadherent erythrocytes has recently been clearly visualized in vivo using polarized light imaging (in the buccal and rectal microcirculations) and by high-resolution fluorescein angiography of the retinal circulation.17,18

different shear rates encountered in the circulation (non-Newtonian behaviour). Only at haematocrits <12% (i.e. severe anaemia) do red blood cell suspensions exhibit Newtonian behaviour. Under experimental conditions changes in haema-tocrit over the range commonly encountered in severe malaria (venous haematocrit, 10–30%; capillary values are lower) have major effects on cytoadherence. Rolling increased fivefold as

Figure 43.5 Two electron micrographs (×4320) showing densely packed parasitized erythrocytes sequestered in cerebral venules of a fatal case of cerebral malaria. Note that even when no intracellular para-site is seen, electron dense deposits are evident on the cell membranes indicating the red cell does contain a parasite, but that its body has been missed in the section. The packing of red cells is much tighter than in normal conditions. (Courtesy of Emsrii Pongponratn.)

A

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Figure 43.6 Uninfected red cells must squeeze past the static rigid, spherical cytoadherent parasitized erythrocytes to maintain flow. This is compromised by the reduced deformability of uninfected red cells in severe malaria and the intererythrocytic adhesive forces that mediate rosetting.

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544 SECTION 9 Protozoan Infections

vascular endothelium. The protuberances are not essential for cytoadherence. A small subpopulation of naturally occurring parasites do not induce surface knobs and parasites can be selected in culture which are knob negative (K-) but still cytoad-here. However, natural parasite isolates are nearly always knob positive (K+). PfEMP-1 proteins protrude from the red cell surface offering several Duffy binding-like (DBL) domains each capable of binding to particular vascular ‘receptors’. Analysis of multiple PfEMP-1 sequences has revealed common antigenic determinants in the DBL-1α domain, a constituent of the so-called ‘head structure’ common to all PfEMP-1 variants, that is involved in the formation of rosettes and in cytoadherence.19 PfEMP-1 expression is greatest in the middle of the asexual cycle. PfEMP1 is an important adhesin and also appears to be a major antigenic determinant for the blood stage parasite, although two other variant proteins encoded by different gene families have been identified – the Rifins and the Surfins. Their function is uncertain.

In addition, proteins expressed only on the younger ring stage infected red cells have also been identified in parasite lines which subsequently develop a chondroitin-sulphate A binding phenotype which could play a role in ring stage cytoadherence.

As in other protozoal parasites the immunodominant surface antigen undergoes antigenic variation to ‘change its coat’ and avoid immune-mediated attack. Each P. falciparum var gene appears to have different rates of switching on and off, with a net result that the infecting parasite population ‘switches’ to a new variant of PfEMP1 at an average rate of about 2% per asexual cycle in culture although this may be considerably higher in vivo. Interestingly, the PfEMP-1 gene expressed shows some dependence on previous variant expression, reflect-ing the effects of host immune response on parasite antigenic variation.

In the chronic phase of untreated infections this antigenic variation results in small waves of parasitaemia approximately every 3 weeks. A protein similar to PfEMP-1 named sequestrin (molecular mass 270 kDa) has been identified on the surface of infected red cells using anti-idiotypic antibodies raised against one of the putative vascular receptors CD36 (see below). The protein MESA may also be partially expressed on the surface of the red cell and has been suggested as a contributor to cytoad-herence. The central role of parasite-derived proteins in cytoad-herence is not accepted by all. It has been suggested that cytoadherence is mediated by altered red cell membrane com-ponents such as a modified form of the red cell cytoskeleton protein band 3 (the major erythrocyte anion transporter, also called Pfalhesin). In culture, most P. falciparum parasites lose the ability to cytoadhere after several cycles of replication. In vivo, cytoadherence may be modulated by the spleen. This has been shown in Saimiri monkeys infected with P. falciparum. Parasitized erythrocytes do not cytoadhere in splenectomized monkeys. Rare patients who have had a splenectomy develop falciparum malaria and in some of these all stages of the parasite are seen in peripheral blood smears.

P. vivax generally does not cytoadhere but recent studies indicate significant adhesion to chondroitin sulphate A – the main receptor for placental cytoadhesion. Plasmodium vivax also has a variant subtelomeric multigene family called vir. Its subcellular localization and function has been unclear although there is recent evidence it might mediate attachment to ICAM-1.

The consequences of microcirculatory obstruction are activation of the vascular endothelium, endothelial dysfunc-tion, together with reduced oxygen and substrate supply, which leads to anaerobic glycolysis, lactic acidosis and cellular dysfunction.

CYTOADHERENCE

Cytoadherence is mediated by several different processes.14 The most important parasite ligands are a family of strain-specific, high-molecular-weight parasite-derived proteins termed P. fal-ciparum erythrocyte membrane protein 1 or PfEMP-1. These variant surface antigen (VSA) proteins (molecular mass 240–260 kDa) are encoded by var genes, a family of ~60 genes distributed in three general locations within the haploid genome: either immediately adjacent to the telomere, close to a telomeric var gene, or in internal clusters. Each parasitized red cell expresses the product of a single gene, a process which is tightly controlled at the transcriptional level and varies between different parasites and different PfEMP-1 genes. PfEMP-1 is transcribed, synthesized and stored within the parasite and, beginning at around 12 hours of development, it is then exported to the surface of the infecting erythrocyte. There it is apposed by an electrostatic interaction through the membrane to a submembranous accretion of parasite-derived knob-associated histidine-rich protein (KAHRP), which is in turn anchored to the red cell via the cytoskeleton protein ankyrin. These accretions cause humps or knobs on the surface of the red cell (Figure 43.7) and these are the points of attachment to

Figure 43.7 Freeze fracture electron micrograph of the membrane of a red cell containing mature P. falciparum showing regularly spaced knobs. (Courtesy of David Ferguson.)

43  Malaria 545

is also the receptor for rhinovirus attachment, appears to be the major cytoadherence receptor in the brain. ICAM-1, but not CD36, is upregulated by cytokines (notably TNFα) and pro-vides a plausible pathological scenario whereby cytokine release enhances cytoadherence. At physiological shear rates (i.e. those likely to be encountered in the human microcirculation) the binding forces (c.10–10N) are similar for CD36 and ICAM-1. For both, the forces of attachment are lower than those required for detachment, which suggests post-attachment alterations to increase adhesion. Binding to the two ligands is synergistic. Thrombospondin (a natural ligand for CD36) will also bind to some parasitized red cells (probably to modified band 3). Other proteins including VCAM-1, PECAM/CD31, E-selectin and the integrin alpha-beta3 have also been shown to bind in some circumstances. P-selectin has been shown to mediate rolling. The relative importance of these molecules and their interac-tions in vivo is still not clear. Chondroitin sulphate A (CSA) appears to be the major receptor for cytoadherence in the pla-centa. Binding to CSA is mediated by a particular PfEMP1 (var2CSA) which gives hope for a specific vaccine against malaria in pregnancy. Thus the placenta selects a parasite sub-population expressing this epitope. Antibodies which inhibit parasitized red cell cytoadherence by binding var2CSA are gen-erally present in multigravidae in endemic areas, but not pri-migravidae which probably explains why the adverse effects of pregnancy on birth weight are greatest in primigravidae.

VASCULAR ENDOTHELIAL LIGANDS

A number of different cell adhesion molecules expressed on the surface of vascular endothelium have been shown to bind para-sitized red cells (Figure 43.8). The interaction between these proteins and the variant surface adhesin of the parasitized red cell is complex. The property of cytoadherence can be studied in vitro with cells expressing the potential ligands on their surface (e.g. human umbilical vein/dermal microvascular or cerebral endothelial cells or transfected COS cells) or with the immobilized purified candidate ligand proteins. Probably the most important of these proteins is the leukocyte differentia-tion antigen CD36; nearly all freshly obtained parasites bind to CD36. Binding is increased at low pH (<7.0) and in the presence of high calcium concentrations. CD36 is constitutionally expressed on vascular endothelium, platelets and monocytes/macrophages but is usually not present on the surface of cere-bral vessels, although it has been suggested that parasitized erythrocytes could bind via CD36 to platelets adherent to cere-bral vascular endothelium. Endothelial activation causes exocy-tosis of intracellular Weibel–Palade bodies, containing bioactive molecules which include von Willebrand factor (vWF) and angiopoietin-2. Ultralong vWF multimers may mediate cytoad-herence and sequestration by binding activated platelets which express CD36. Concentrations of ADAMTS13, which cleaves and inactivates UL-vWF, are low in patients with severe malaria. The intercellular adhesion molecule (ICAM-1 or CD54), which

Figure 43.8 Schematic representation of cytoadherence in falciparum malaria. On the red cell side (above) the principal ligand is the variant antigen Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This is expressed on the surface of ‘knobs’ which protrude from the red cell surface. It is anchored beneath to the knob-associated histidine-rich protein (KAHRP) and stabilized by PfEMP3. The rifin and CLAG gene products are not directly involved in adhesion but CLAG does appear to be required for cytoadherence. Parasite-modified band 3 (the major anion transporter) contributes to adhesion probably by binding to thrombospondin (TSA). Sequestrin is a distinct parasite-derived protein also mediating adhesion. The ring stage adhesion (not shown) is distinct from PfEMP1 and expressed in the first third of the asexual cycle. On the vascular endo-thelial side many molecules facilitate adhesion by binding PfEMP1. The most important is the cellular differentiation antigen: CD36. Intercellular adhesion molecule 1 (ICAM1) is important particularly in the brain, elsewhere it synergizes with CD36. Chondroitin sulphate A (CSA) attached to thrombomodulin (TM) is very important for placental sequestration. The other identified adhesion molecules are vascular cell adhesion molecule 1 (VCAM1), E-selectin, platelet endothelial cell adhesion molecule 1 (PECAM1), αβ3 integrin, heparan sulphate (HS) and P-selectin.

Parasitized erythrocyte

Host cell

CLAG

ModifiedBand 3

TM

P-selectin

HS CSA TSA CD36 ICAM1

PECAM1 VCAM1

HAPf EMP1

E-selectin

αβ3

KAHRP Pf EMP3Rifin

Sequestrin

546 SECTION 9 Protozoan Infections

RED CELL DEFORMABILITY

As Plasmodium vivax matures inside the erythrocyte, the cell enlarges and becomes more deformable. Plasmodium falci-parum does exactly the opposite; the normally flexible bicon-cave disc becomes progressively more spherical and rigid. The reduction in deformability results from reduced membrane flu-idity, increasing sphericity and the enlarging and relatively rigid intraerythrocytic parasite. Infected red cells are less filterable than uninfected cells. It has been argued that sequestration is an adaptive response to escape splenic filtration. However, reduced deformability alone cannot account for microvascular obstruction as it would lead to obstruction at the mid-capillary (i.e. the smallest internal diameter in the vasculature) and could not explain sequestration in venules. Loss of uninfected red cell deformability has been recognized as a major contributor to disease severity and outcome. Increased erythrocyte rigidity measured at the low shear stresses encountered in capillaries and venules is correlated closely with outcome in severe malaria,

Other as yet unidentified vascular receptors are also present, as sequestration also occurs in vessels expressing none of the potential ligands identified so far. Thus ICAM-1 appears to be the major vascular ligand in the brain involved in cerebral sequestration, CSA is the major ligand in the placenta, and CD36 is probably the major ligand in the other organs. The relationship between cytoadherence, measured ex vivo and the severity of infection or clinical manifestations has been incon-sistent between studies. This is not particularly surprising as all parasitized erythrocytes cytoadhere. Severity is related to the number of parasites in the body and the distribution of cytoad-herence within the vital organs. The relative importance of parasite phenotype and the various potential vascular ligands in the pathophysiology of severe falciparum malaria and the precise role of the spleen still remains to be determined.

ROSETTING

Erythrocytes containing mature parasites also adhere to unin-fected erythrocytes. This process leads to the formation of ‘rosettes’ when suspensions of parasitized erythrocytes are viewed under the microscope (Figure 43.9). Rosetting shares some characteristics of cytoadherence. It starts at around 16 hours of asexual life cycle development (slightly after cytoad-herence begins) and it is trypsin-sensitive. But parasite species which do not sequester do rosette and unlike cytoadherence, rosetting is inhibited by certain heparin subfractions and calcium chelators. Furthermore, whereas all fresh isolates of P. falciparum cytoadhere, not all rosette. Rosetting is mediated by attachment of a specific subgroup of PfEMP1 adhesins (with red cell binding mediated by the N-terminal DBL1α1 domain) to the complement receptor CR1, heparan sulphate, blood group A antigen and probably other red cell surface molecules. Attach-ment is facilitated by serum components including Comple-ment factor D, albumin and IgG anti-band 3 antibodies. The protective effect of group O against severe malaria is thought to result from reduced rosetting. The forces required to separate a rosette are approximately five times greater than those required to separate cytoadherent cells, although shearing forces may still be effective in disrupting rosettes in vivo. When known rosetting parasite lines (K+R+) were perfused through the rat mesocae-cum, an ex vivo model for the study of vascular perfusion, they caused significantly more microvascular obstruction than iso-lates which cytoadhered but did not rosette (K+R−). Rosetting has been associated with severe malaria in some studies but not in others. It has been suggested that rosetting might encourage cytoadherence by reducing flow (shear rate), which would enhance anaerobic glycolysis, reduce pH and facilitate adher-ence of infected erythrocytes to venular endothelium. Rosetting tends to start in venules and this could certainly reduce flow. The adhesive forces involved in rosetting could also impede forward flow of uninfected erythrocytes as they squeeze past sticky cytoadherent parasitized red cells in capillaries and venules (Figure 43.6). The mechanical obstruction or ‘static hindrance’ would be compounded by the lack of deformability of the adher-ent parasitized red cells and circulating unparasitised red cells.

AGGREGATION

Platelet-mediated aggregation of parasitized erythrocytes is mediated via platelet CD36 and is associated with disease sever-ity. Aggregation could also contribute to vascular occlusion.

Figure 43.9 Rosetting. (A) Uninfected red blood cells bind to a P. vivax-infected erythrocyte. (B) Transmission electron micrograph of a rosette around a P. falciparum-infected erythrocyte. (A, Courtesy of Rachanee Udomsangpetch. B, Courtesy of David Ferguson.)

A

B

43  Malaria 547

of a specific cellular immune responses and immune memory. In severe malaria there is evidence of a broader immune sup-pression, with defects in monocyte and neutrophil chemotaxis, reduced neutrophil and monocytic phagocytic function (which may result from ingestion of malaria pigment), and a tendency to bacterial super-infection. In the nephrotic syndrome associ-ated with chronic P. malariae infections, malaria antigen and immune complexes can be eluted from the kidney, indicating an immunopathological progress in this condition. But why some children are affected but the majority are not remains unresolved.

VASCULAR PERMEABILITY

There is evidence of a mild generalized increase in systemic vascular permeability in severe malaria. Focal perivascular and intraparenchymal oedema is seen in the brain in 70% of fatal cases. In the past it was suggested that cerebral malaria resulted from a marked increase in cerebral capillary permeability, which led to brain swelling, coma and death. The imaging studies conducted to date indicate that, although there may be some increases in brain water, as might be expected given the wide-spread venular and capillary obstruction, and some patients do develop cerebral oedema (particularly agonally), the majority of adults and children with cerebral malaria do not have sub-stantial cerebral oedema (Figure 43.10). However, the role of raised intracranial pressure in cerebral malaria still remains unclear. Whereas 80% of adults have opening pressures at lumbar puncture which are in the normal range (<200 mm CSF), 80% of children have elevated opening pressures (>100 mm CSF: the normal range is lower in children) and intracranial pressure may rise transiently to very high levels. Uncontrolled epileptic seizure activity increases cerebral metab-olism thereby increasing the imbalance between energy demand and limited supply (because of microvascular obstruction) and may cause brain swelling. Some patients with cerebral malaria die from acute respiratory arrest with neurological signs that are compatible with brain stem compression. But these signs are also common and may persist for many hours in survivors. The elevation in CSF opening pressure is usually not great (in general, it is much lower than in bacterial or fungal meningitis) and there is no difference between these lumbar puncture opening pressures in surviving children and fatal cases. Studies of computerized tomography (CT) or magnetic resonance imaging (MRI) have generally shown brain swelling in cerebral malaria (compatible with an increased intracerebral blood volume resulting from sequestration), sometimes discrete focal areas of oedema (particularly in white matter) or abnor-mal areas of signal attenuation in severe cases, but usually not generalized cerebral oedema. MRI studies show abnormal T2 signal intensity; and diffusion-weighted abnormalities in the cortical, deep gray and white matter structures. Focal abnor-malities evident on MRI do not conform to arterial vascular distributions.21

Where generalized cerebral oedema has been reported it has sometimes been inferred from brain swelling on CT and could have resulted from increased intracerebral blood volume. Immunohistochemical studies on autopsy brain tissues indicate focal disruption of specialized endothelial cell tight junctions and endothelial activation in areas of intense sequestration, but clinical investigations have also failed to detect major alterations in blood–brain barrier permeability. Thus, raised intracranial

and when assessed at the shear rates encountered on the arterial side and importantly, in the spleen, reduced red cell deform-ability also correlates with anaemia.

IMMUNOLOGICAL PROCESSES

The contribution of immune processes to the pathology of human malaria remains uncertain. It has been suggested that severe malaria, and in particular cerebral malaria, results from specific immune-mediated damage. This is unlikely. Confusion arises when the term cerebral malaria is applied equally to human disease and to neurological dysfunction in animal models infected with unusual parasites. Neuropathology in rodent models does result from immune-mediated damage, but human cerebral malaria has very different pathology and very different responses to interventions. In rapidly fatal cerebral malaria there is intense parasitized red cell sequestration, but relatively few leukocytes are found in or around the cerebral vessels in fatal cases, although recent pathological studies have shown more host leukocyte and particularly platelet accumula-tion in the cerebral vasculature of African children who died from cerebral malaria compared to the findings in South-east Asian adults.16,20 The degree of host leukocyte response depends on the stage of infection and is less than that seen in other organs such as the kidney or lung, which may relate to the immunologically privileged state of the cerebral parenchyma. When leukocytes are seen they are often fulfilling their house-keeping role of clearing away residual cytoadherent membranes and pigment. There is little pathological evidence in man for widespread cerebral vasculitis in cerebral malaria although there is undoubted endothelial activation, and recent studies have shown evidence for intraparenchymal responses including widespread astroglial activation, evidence of blood–brain barrier leakage, and axonal injury.

Although some glomerular abnormalities have been noted in fatal malaria the clinical and pathological findings suggest that acute tubular necrosis, and not acute glomerulonephritis, is the cause of renal dysfunction. The pathogenesis of pulmo-nary oedema is uncertain – as it is for the adult respiratory distress syndrome in other conditions – but it is unlikely to involve a specific immune-mediated process. Thus despite the enormous intravascular antigenic load in malaria, with the for-mation and deposition of immune complexes and variable complement depletion, there is little evidence of a specific immunopathological process in severe falciparum malaria.

While innate immune responses are very important in con-trolling malaria, acute infections are associated with malaria antigen-specific unresponsiveness. This selective paresis is one of the factors contributing to the slow development of an effec-tive and specific immune response in malaria. Acute malaria is characterized by non-specific polyclonal B-cell activation. There is a reduction in circulating T cells with an increase in the γ/δ T-cell subset, but other T-cell proportions are usually normal. Although residents of hyperendemic or holoendemic malarious areas have hypergammaglobulinaemia, most of this antibody is not directed against malaria antigens. In non-immune indi-viduals, the acute antibody response to infection often com-prises mostly IgM or IgG2, isotypes which are unable to arm cytotoxic cells and thus kill asexual malaria parasites. These observations have led to the suggestion that malaria induces an immunological ‘smoke-screen’ with broad-spectrum and non-specific activation that interferes with the orderly development

548 SECTION 9 Protozoan Infections

Local release of haemoglobin and haem depletes nitric oxide (NO) causing endothelial dysfunction. L-arginine (an NO pre-cursor) concentrations are low and asymmetric dimethylargi-nine levels (an inhibitor of NO synthase) are increased in patients with severe malaria. Reversible axonal dysfunction has recently been shown in neuropathological studies and this probably plays an important role in central nervous system dysfunction. Coma in malaria is not caused by raised intracra-nial pressure. There has been considerable interest in the mech-anism of coma and attempts to reverse it. But it should be remembered that the brain in cerebral malaria has a compro-mised blood supply and waking a patient from coma may result in an increased cerebral metabolic demand. Coma in cerebral malaria could be neuroprotective.

ACUTE KIDNEY INJURY

There is renal cortical vasoconstriction and consequent hypo-perfusion in severe falciparum malaria. In patients with acute kidney injury (AKI) renal vascular resistance is increased. The renal injury in severe malaria results from acute tubular necro-sis. The oxygen consumption of the kidneys is reduced in AKI and it is not improved by dopamine-induced arteriolar vasodi-latation and consequent increase in renal blood flow suggesting a fixed injury. Acute tubular necrosis presumably results from renal microvascular obstruction and cellular injury consequent upon sequestration in the kidney and the filtration of nephro-toxins such as free haemoglobin, myoglobin and other cellular material. AKI always recovers fully in survivors. Significant glo-merulonephritis is very rare. The role of systemic and local cytokine release and altered regulation of renal microvascular flow is uncertain. Massive haemolysis compounds the insult in blackwater fever complicating malaria and haemoglobinuria may itself lead to renal impairment. AKI also occurs in young children with severe malaria and levels of blood urea are impor-tant independent prognostic indices, but established acute renal failure requiring renal replacement is almost exclusively con-fined to older children and adults.

pressure probably arises mainly from an increase in cerebral blood volume. Any contribution of increased permeability is likely to be small. The increased cerebral blood volume results from the addition of the circulating blood required to maintain cerebral perfusion and the considerable sequestered static biomass of intracerebral parasitized erythrocytes. Children may be particularly vulnerable as after the skull sutures have fused, as there is less space for cranial expansion than in adults. The relationship between intracerebral pressure and volume is non-linear (i.e. once the brain has expanded to fill the skull, only small further increases in volume cause large increases in intra-cranial pressure). The possibility that a sudden rise in intracra-nial pressure accounts for some deaths cannot be excluded.

PATHOGENESIS OF COMA

Coma in severe malaria is called cerebral malaria. Although several factors may contribute to impaired consciousness in severe malaria (seizures, hypoglycaemia) there is a syndrome of diffuse but reversible encephalopathy which is characteristic of malaria and is not seen in other infections. The cause of coma is not known. There is undoubtedly an increase in cerebral anaerobic glycolysis with cerebral blood flows that are inap-propriately low for the arterial oxygen content, increased cere-bral metabolic rates for lactate and increased CSF concentrations of lactate, but these changes which reflect impaired perfusion, do not provide sufficient explanation for coma. Presumably the metabolic milieu created adjacent to the sequestered and highly metabolically active parasites and their attachment to the acti-vated cerebral vascular endothelium interferes with endothelial and blood–brain barrier function. But how this then interferes with neurotransmission is not known. Cytokines increase pro-duction of nitric oxide, a potent inhibitor of neurotransmis-sion, by leukocytes, smooth muscle cells, microglia and vascular endothelium through induction of the enzyme nitric oxide syn-thase. Inducible nitric oxide synthase expression is increased in the brain in fatal cerebral malaria. Thus, local synthesis of nitric oxide could be relevant to the impairment of consciousness.

Figure 43.10 (A) T1-weighted magnetic resonance imaging (550/25 TR/TE) of the brain in a 28-year-old man with cerebral malaria. (B) Following recovery of consciousness showing shrinkage. (C) 135 days later the brain is normal. There was no evidence of cerebral oedema on T2-weighted images. The acute swelling was interpreted as representing increased intracerebral blood volume.

Day 0 Day 3 Day 135

A B C

43  Malaria 549

spleen, with the degree of resulting anaemia. The mechanism responsible has not been identified, although there is evidence in acute malaria for increased oxidative damage which might compromise red cell membrane function and deformability.23 In simian malarias there is evidence of an inversion of the erythrocyte membrane lipid bilayer in uninfected erythrocytes, but this has not been studied in man. The role of antibody (i.e. Coombs’-positive haemolysis) in anaemia is unresolved. The majority of studies to date do not show increased red cell immunoglobulin binding in malaria, but in the presence of a lowered recognition threshold for splenic clearance, this might be difficult to detect. The splenic threshold for the clearance of abnormal erythrocytes, whether because of anti-body coating or reduced deformability, is lowered.23 Thus, the spleen removes large numbers of relatively rigid cells causing shortened erythrocyte survival, particularly in severe malaria. This is unaffected by corticosteroids. The spleen also fulfils its normative function of removing damaged intraerythrocytic parasites from red cells (particularly following treatment with an artemisinin derivative and returning the ‘once parasitized’ red cells back to the circulation by a process of ‘pitting’.23 These erythrocytes then have reduced survival and so following suc-cessful treatment of hyperparasitaemia may result in a delayed haemolytic anaemia.

In the context of acute uncomplicated malaria the anaemia is worse in younger children and those with protracted infec-tions. Loss of unparasitized erythrocytes accounts for approxi-mately 90% of the acute anaemia resulting from a single uncomplicated infection. Iron deficiency and malaria often coincide in the same patient and in some areas routine iron supplementation following malaria promotes recovery from anaemia.

COAGULOPATHY AND THROMBOCYTOPENIA

There is accelerated coagulation cascade activity with acceler-ated fibrinogen turnover, consumption of antithrombin III, reduced factor XIII and increased concentrations of fibrin deg-radation products in acute malaria. In severe infections, the antithrombin III, protein S and protein C are further reduced and prothrombin and partial thromboplastin times may be pro-longed. In occasional patients (<5%), bleeding may be signifi-cant. The coagulation cascade is activated via the intrinsic pathway. Intravascular thrombus formation is observed rarely at autopsy in fatal cases and fibrin deposition is sparse and platelets are strikingly unusual in adults, in contrast to paediat-ric cases.

Thrombocytopenia is common to all the human malarias and is caused by increased splenic clearance. Thrombocytope-nia is associated with high levels of IL-10 and appropriately raised concentrations of thrombopoietin (a key growth factor for platelet production). Plasma concentrations of macrophage colony stimulating factor are high, which stimulate macrophage activity and may increase platelet destruction. Platelet turnover is increased. The role of platelet-bound antibody in malarial thrombocytopenia is controversial. There has been evidence of platelet activation in some studies, but not others. Erythrocytes containing mature parasites may activate the coagulation cascade directly and cytokine release is also procoagulant. The high plasma levels of P-selectin found in severe malaria may derive from platelets, but could also come from vascular

PULMONARY OEDEMA

Despite intense sequestration in the myocardial vessels, the heart’s pump function is remarkably well preserved in severe malaria. Pulmonary oedema in falciparum, vivax and knowlesi malaria results from a sudden increase in pulmonary capillary permeability that is not reflected in other vascular beds. The pulmonary capillary wedge pressure is usually normal and the pressure threshold for the development of pulmonary oedema is relatively low. The cause of this increase in pulmonary capil-lary permeability is not known, although the presence of sequestered PRBC and host leukocytes in pulmonary capillaries may have a role in causing pulmonary capillary endothelial cell dysfunction.

FLUID SPACE AND ELECTROLYTE CHANGES

Haemodynamic studies in adults clearly point to microvascular and not macrovascular dysfunction as the primary circulatory abnormality in severe malaria. Following rehydration the plasma volume is increased in moderate and severe malaria. In most adults total body water and extracellular volume are normal. Plasma renin activity, aldosterone and antidiuretic hormone concentrations are elevated, reflecting an appropriate activation of homeostatic mechanisms to maintain adequate circulating volume in the presence of general vasodilatation and a falling haematocrit. Mild hyponatraemia and hypochloraemia are common in severe malaria, but serum potassium concentra-tions are usually normal. Occasionally hyponatraemia is severe. Studies in Kenyan children indicate inappropriate antidiuretic hormone (arginine vasopressin) secretion in two-thirds of cases. There has been much recent debate whether children with severe malaria are hypovolaemic and fluid-depleted. When measured directly in Gabonese children total body water was either normal or slightly reduced arguing against dehydration, whereas studies in Kenyan children suggested there may be benefit from infusions of colloid, particularly albumin solu-tions. However a definitive multicentre study in African chil-dren was stopped early because of increased mortality in those receiving vigorous crystalloid or albumin fluid loading.22

ANAEMIA

The pathogenesis of anaemia is multifactorial. It results from the obligatory destruction of red cells containing parasites at merogony, the accelerated destruction of non-parasitized red cells that parallels disease severity, and it is compounded by bone marrow dyserythropoiesis. In severe malaria anaemia develops rapidly; the rapid haemolysis of unparasitized red cells is the major contributor to the decline in haematocrit. Bone marrow dyserythropoiesis persists for days or weeks following acute malaria and reticulocyte counts are usually low in the acute phase of the disease. The cause of the dyserythro-poiesis is thought to be related to intramedullary cytokine pro-duction. Serum erythropoietin levels are usually elevated, although in some series it has been suggested that the degree of elevation was not sufficient for the degree of anaemia. In falciparum malaria the entire red cell population (i.e. both infected and uninfected red cells) becomes more rigid. This loss of deformability correlates with disease severity and outcome and, when measured at the high shear rates encountered in the

550 SECTION 9 Protozoan Infections

THE SPLEEN

There is considerable and rapid splenic enlargement in malaria, mainly as a result of cellular multiplication and structural change and an enhanced capacity to clear red cells from the circulation both by Fc receptor-mediated (immune) mecha-nisms and by recognition of reduced deformability (filtra-tion).23 The increased filtration of the spleen and the reduced deformability of the entire red cell population results in the rapid development of anaemia in severe malaria. The spleen may also modulate cytoadherence. It plays a central role in limiting the acute expansion of the malaria infection by remov-ing parasitized erythrocytes and this has led to the suggestion that a failure to augment splenic clearance sufficiently rapidly may be a factor in the development of severe malaria. Charac-teristic changes to the immuno-architecture of the spleen are seen during infection which may reflect a central role for den-dritic cells in orchestrating specific immune responses.

The spleen is capable of removing damaged intraerythro-cytic parasites and returning the once infected red cells to the circulation (a process known as ‘pitting’), where they have shortened survival. This is an important contributor to parasite clearance following antimalarial drug treatment (particularly treatment with artemisinin derivatives).

GASTROINTESTINAL DYSFUNCTION

Abdominal pain may be prominent in acute malaria. Minor stress ulceration of the stomach and duodenum is common in severe malaria. The pattern of malabsorption of sugars, fats and amino acids suggests reduced splanchnic perfusion. This results from both gut sequestration and visceral vasoconstriction. Gut permeability is increased and this may be associated with reduced local defences against bacterial toxins, or even whole bacteria in severe disease. Antimalarial drug absorption is remarkably unaffected in uncomplicated malaria, except for those drugs which have fat- (i.e. food-) dependent absorption (atovaquone, lumefantrine).

LIVER DYSFUNCTION

Jaundice is common in adults with severe malaria and there is other evidence of hepatic dysfunction, with reduced clotting factor synthesis, reduced metabolic clearance of the antimalarial drugs and a failure of gluconeogenesis which contributes to lactic acidosis and hypoglycaemia. Nevertheless, true liver failure (as in fulminant viral hepatitis) does not occur. There is seques-tration in the hepatic microvasculature and, although many patients with acute falciparum malaria have elevated liver blood flow values, in very severe infections liver blood flow is reduced. In adults, liver blood flow values <15 mL/kg per minute are associated with elevated venous lactate concentrations, which suggests a flow limitation to lactate clearance and thus a contri-bution of liver dysfunction to lactic acidosis. Direct measure-ments of hepatic venous lactate concentrations in severe malaria confirm that the hepatosplanchnic extraction ratio is inversely correlated with mixed venous plasma lactate (i.e. hyperlactatae-mia is associated with reduced liver clearance of lactate). There is no relationship between liver blood flow and impairment of antimalarial drug clearance. Jaundice in malaria appears to have haemolytic, hepatic and cholestatic components. Cholestatic jaundice may persist well into the recovery period. There is no residual liver damage following malaria.

endothelium, as plasma concentrations of other endothelial-derived proteins (thrombomodulin, E-selectin, ICAM-1, VCAM-1) are also elevated. It was suggested in the past that disseminated intravascular coagulation (DIC) is important in the pathogenesis of severe malaria, but detailed prospective clinical and pathogenesis studies have refuted this. Coagulation cascade activity is directly proportional to disease severity, but hypofibrinogenaemia resulting from DIC is significant in less than 5% of patients with severe malaria and lethal haemorrhage (usually gastrointestinal) is very unusual.

BLACKWATER FEVER

This is a poorly understood condition (Figure 43.11), in which there is massive intravascular haemolysis and the passage of ‘Coca-Cola’-coloured urine. Historically, this was linked to fre-quent quinine self-medication in expatriates living in malarious areas and indeed blackwater fever almost disappeared from Africa during the ‘chloroquine’ era from 1950 to 1980 but has since reappeared. Blackwater (urine) occurs in four circum-stances: (1) when patients with G6PD deficiency take oxidant drugs (e.g. primaquine or sulphones), irrespective of whether they have malaria or not; (2) occasionally when patients with G6PD deficiency have malaria and receive quinine treatment; and (3) in some patients with severe falciparum malaria who have normal erythrocyte G6PD levels irrespective of the treat-ment given (4) when people who are exposed to malaria self-medicate frequently with quinine (or structurally related drugs). In severe malaria, rates of blackwater in Asian patients are similar whether the patients receive quinine or an artemisinin derivative. How quinine causes blackwater in these last three situations is not known, as it is not an oxidant drug. G6PD-deficient red cells are particularly susceptible to oxidant stress as they are unable to synthesize adequate quantities of NADPH through the pentose shunt. This leads to low intraerythrocytic levels of reduced glutathione and catalase and consequent alter-ations in the erythrocyte membrane and increased susceptibil-ity to organic peroxides. Blackwater fever may be associated with acute renal failure, although in the majority of cases renal function remains normal.

Figure 43.11 Blackwater fever and cerebral malaria. A 22-year-old male with severe malaria and massive haemolysis.

43  Malaria 551

(the Pasteur effect), the increased metabolic demands of the febrile illness, and the obligatory demands of the parasites which use glucose as their major fuel (all of which increase demand); and a failure of hepatic gluconeogenesis and glycoge-nolysis (reduced supply). Hepatic glycogen is exhausted rapidly: stores in fasting adults last approximately 2 days, but children only have enough for 12 hours. Healthy children have approxi-mately three times higher rates of glucose turnover compared with adults, but in severe malaria turnover is increased by more than 50% (to values five times higher than those in adults with severe malaria). The net result of impaired gluconeogenesis, limited glycogen stores and greatly increased demand results in a hypoglycaemia in 20–30% of children with severe malaria. In patients treated with quinine, this is compounded by quinine-stimulated pancreatic β-cell insulin secretion. Hyperinsulinae-mia is balanced by a reduced tissue sensitivity to insulin, which returns to normal as the patient improves. This probably explains why quinine-induced (hyperinsulinaemic) hypogly-caemia tends to occur after the first 24 hours of treatment, whereas malaria-related hypoglycaemia (with appropriate sup-pression of insulin secretion) is often present when the patient with severe malaria is first admitted. Hypoglycaemia contrib-utes to nervous system dysfunction and in cerebral malaria is associated with residual neurological deficit in survivors.

PLACENTAL DYSFUNCTION

Pregnancy increases susceptibility to malaria. This is probably caused by a suppression of systemic and placental cell-mediated immune responses. There is intense sequestration of P. falci-parum-infected erythrocytes in the placenta, local activation of pro-inflammatory cytokine production and maternal anaemia. This leads to cellular infiltration and thickening of the syncytiotrophoblast and placental insufficiency with conse-quent fetal growth retardation. Illness close to term also results in prematurity. In areas of intense transmission a malaria attributable reduction in birth weight (circa 170 g) is confined to primigravidae. There is no convincing evidence that malaria causes abortion or stillbirth in this context. With lower levels of transmission (i.e. less immunity) the risk extends to other pregnancies and there is a propensity to develop severe malaria with a high incidence of fetal death. Plasmodium vivax also reduces birth weight (by about two-thirds the amount caused by P. falciparum), which questions the primary role of extensive sequestration in the pathogenesis of placental insufficiency. Malaria in early pregnancy may cause abortion.

BACTERIAL INFECTION

Patients with severe malaria are vulnerable to bacterial infec-tions, particularly of the lungs and urinary tract (following catheterization). Postpartum sepsis is also common. Spontane-ous bacterial septicaemia is an important complication of severe malaria. This is relatively unusual in adults (probably <1% of cases) but is much more common in young children. There is undoubtedly considerable overlap between sepsis (both pneumonia and septicaemia) and malaria in endemic areas. The difficulty is one of diagnosis; where transmission is high and parasitaemia is common in children, it may be difficult or impossible to distinguish bacterial infections with coincident parasitaemia from infections complicating malaria. The blood

ACIDOSIS

Acidosis is a major cause of death in severe falciparum malaria both in adults and children. This has been considered to be mainly a lactic acidosis, although ketoacidosis (and sometimes salicylate intoxication) may predominate in children and the acidosis of renal failure is common in adults. In severe malaria the arterial, capillary, venous and CSF concentrations of lactate rise in direct proportion to disease severity. Acid–base assess-ment or venous lactate concentrations four hours after admis-sion to hospital are very good indicators of prognosis in severe malaria. In bacterial sepsis, there is also hyperlactataemia, but, unless there is profound shock, the lactate–pyruvate ratio is usually less than 15. This indicates that hypermetabolism is the source of lactate accumulation. In severe malaria the pathogen-esis is different; lactate–pyruvate ratios often exceed 30 reflect-ing tissue hypoxia and anaerobic glycolysis. Lactic acidosis results from several discrete processes: the tissue anaerobic gly-colysis consequent upon microvascular obstruction; a failure of hepatic and renal lactate clearance; and the production of lactate by the parasite. Hypovolaemia is usually not a major contributor to lactic acidosis. Mature malaria parasites consume up to 70 times as much glucose as uninfected cells and over 90% of this is converted to L+lactic acid (plasmodia do not have the complete set of enzymes necessary for the citric acid cycle). Interestingly, up to 6% of the lactic acid appears as D-lactate, but this does not contribute materially to the acidosis. However calculations based on glucose and lactate turnover in man indi-cate that the majority of the lactic acid produced in malaria derives from host rather than parasite sources. Lactate levels also rise after generalized convulsions. Lactate turnover in both adults and children with severe malaria is increased approxi-mately three-fold compared with values obtained in healthy adults. Studies in children using stable isotope techniques indi-cate that increased lactate production (resulting from anaerobic glycolysis) rather than reduced clearance is the main cause of lactate accumulation, although in adults reduced clearance is certainly a contributor. Hyperlactataemia is associated with hypoglycaemia and is accompanied by hyperalaninaemia and elevated glycerol concentrations reflecting the impairment of gluconeogenesis through the Cori cycle. Lactate, glutamine and alanine are the major gluconeogenic precursors. There is evi-dence for the presence of another, as yet unidentified strong organic anion, in acidotic patients with severe malaria, which is the major contributor to acidosis.

Triglyceride and free fatty acid levels are also elevated in acute malaria and plasma concentrations of ketone bodies are raised in patients who have been unable to eat. Ketoacidosis may be prominent in children. In severe malaria, there is dys-function of all organ systems, particularly those with obligatory high metabolic rates. The endocrine glands are no exception. Pituitary–thyroid axis abnormalities result in the ‘sick euthy-roid’ syndrome and also parathyroid dysfunction. Mild hypo-calcaemia is common and hypophosphataemia may be profound in the very seriously ill. By contrast, the pituitary–adrenal axis appears normal in acute malaria.

HYPOGLYCAEMIA

Hypoglycaemia is associated with hyperlactataemia and shares the same pathophysiological aetiology: an increased peripheral requirement for glucose consequent upon anaerobic glycolysis

552 SECTION 9 Protozoan Infections

are only a few, or no parasites in the cerebral vessels. This occurs when death was several days after parasite clearance, or the diagnosis was incorrect. Secondary neuropathological changes include widespread astroglial activation and nonspecific signs of neuronal stress response. Axonal injury and consequent dys-function correlate with premortem coma and this provides a plausible mechanism whereby the parasitized erythrocyte, remaining within the vascular space, can reversibly affect neu-rological function.

HEART AND LUNGS

Despite intense sequestration in the myocardial microvascula-ture the heart is remarkably normal, although petechial epicar-dial haemorrhages are common and in anaemic patients the heart is commonly pale and dilated. As in all other organs, extravascular pathological changes are rare. In adults the lungs often show evidence of pulmonary oedema, although this may be patchy. Hyaline membrane formation suggests leakage of proteinaceous fluid. There is moderate sequestration and leu-kocyte aggregates are more prominent than in the brain. There may be secondary bacterial pneumonia. Pathophysiological studies in P. vivax infections suggest pulmonary vascular seques-tration but as yet there is no pathological confirmation of this.

LIVER AND SPLEEN

The liver is generally enlarged and may be black from malaria pigment. There is congestion of the centrilobular capillaries with sinusoidal dilatation and Kupffer cell hyperplasia. Seques-tration of parasitized erythrocytes is associated with variable cloudy swelling of the hepatocytes and perivenous ischaemic changes, and sometimes centrizonal necrosis. In adults, hepatic glycogen is often present despite hypoglycaemia. In uncompli-cated malaria the liver histology is often normal. The spleen is often dark or black from malaria pigment, enlarged, soft and friable. It is full of erythrocytes containing mature and imma-ture parasites. There is evidence of reticular hyperplasia and architectural reorganization. The soft and acutely enlarged spleen of acute lethal infections contrasts with the hard fibrous enlargement associated with repeated malaria.

KIDNEYS

The kidneys are often slightly swollen. In adults there are com-monly tubular abnormalities consistent with ischaemia, includ-ing acute tubular necrosis and tubular epithelial cell regenerative change. There is patchy sequestration, particularly in the glo-merular capillaries, although this is less than that seen in cere-bral capillaries. Occasional mesangial and endothelial cell proliferative changes are seen. Leukocyte sequestration is similar to that in the lung and more marked than in the brain. Immu-nofluorescence and electron microscopic studies show minimal immunoglobulin deposition on the glomerular capillary base-ment membranes, but the changes are not those of a primary immune complex-mediated glomerulonephritis.

ALIMENTARY TRACT

Upper gastrointestinal bleeding from erosions may occur in severe malaria. There is intense sequestration in the gut and visceral ischaemia may explain the acute abdominal pain that

smear is sensitive but not specific for malaria as the cause of the illness, whereas blood culture is insensitive in the diagnosis of bacteraemia. Recent studies suggest that plasma Pf HRP2 con-centrations may be a useful discriminant. Non-typhoid Salmo-nella septicaemias are an important complication of otherwise uncomplicated falciparum malaria in African children. Malaria predisposes young children to systemic non-typhoidal Salmo-nella infections which are a major cause of septicaemia in sub-Saharan Africa, particularly where HIV-AIDS is prevalent.

HistopathologyAs the benign human malarias are rarely fatal there is relatively little information available on the histopathology of these infec-tions. Unfortunately, this is not the case for P. falciparum malaria. In fatal malaria, the microvasculature of the vital organs is packed with erythrocytes containing mature forms of the parasite. There is abundant intra- and extra-erythrocytic pigment and organs such as the liver, spleen and placenta may be grey-black in colour. Sequestration is not uniformly distrib-uted; it tends to be greatest in the brain and heart followed by the gut, kidney, adipose tissue, liver, lungs and least of all in bone marrow and skin. There is remarkably little extravascular pathology in malaria.

BRAIN

If the patient dies from the acute infection, the brain is com-monly mildly swollen with multiple small petechial haemor-rhages throughout the white matter. Different architectural types of haemorrhage are seen; simple petechial, zonal ring haemorrhages, and Durck’s granulomata. Haemorrhages are less prominent in the grey matter. Large haemorrhages or infarcts are rare. There is usually no evidence of tentorial or foramen magnum herniation. Capillaries and venules are dis-tended and packed with erythrocytes containing mature forms of the parasite (whereas these are seen rarely in peripheral blood smears) (Figure 43.5). This sequestration is particularly promi-nent in the white matter, although the tissue is much less vas-cular than the grey matter. The degree of cerebral sequestration and the intensity of erythrocyte packing is greater in cerebral malaria than in fatal malaria in which the patient was not coma-tose. A large quantity of intra- and extra-erythrocytic pigment is evident. In the white matter, accumulations of glial cells are seen surrounding haemorrhagic foci (Durck’s granuloma) where vessels appear to have been occluded by a mass of parasit-ized cells and then ruptured. At a microvascular level, there is considerable variation in the intensity of sequestration between vessels, with each vessel having a discrete age distribution of parasite maturity. At the ultrastructural level the erythrocytes are seen to be packed closely together and the infected red cells are adherent to the vascular endothelium by attachment of knob-like surface projections to the endothelial surface. In adults occasional fibrin strands are seen but there is a striking absence of platelets and usually only focal leukocyte aggrega-tion, i.e. there is no evidence of thrombus formation or vascu-litis. In children there is more fibrin deposition and platelets can be seen. On immunofluorescent staining malarial antigens may be seen on the endothelial basement membrane, but the significance of this observation is uncertain (i.e. does it reflect pathology in vivo or an agonal artefact?). In some cases there

43  Malaria 553

thickening, macrophage infiltration, pigment deposition and perivillous fibrin deposition. Active infection is associated with basement, membrane thickening, fibrinoid necrosis and syncy-tial knots. Chronic infection is associated with marked mono-nuclear cell infiltration.

Laboratory DiagnosisMalaria is diagnosed by microscopic examination of the blood. It is not a clinical diagnosis.

Thick and thin blood films are made on clean, grease-free glass slides (Figure 43.12). Having written the patient’s name, time and date, the glass slide can be cleaned by breathing on the surface and wiping with a clean cloth. The patient’s finger should be cleaned with alcohol, allowed to dry and then the side of the finger tip should be pricked with a sharp sterile lancet or

sometimes occurs in severe malaria. Despite this, drug absorp-tion is often remarkably normal.

BONE MARROW

Dyserythropoietic change is prominent in all the acute malarias. Bone marrow macrophages contain pigment and erythropha-gocytosis may be seen. Iron is usually plentiful. The platelet and white cell series are usually normal.

PLACENTA

The placenta may be black from malaria pigment even if the mother is asymptomatic throughout pregnancy. Large numbers of mature P. falciparum parasites are seen on crush smears, although the peripheral blood smear may be negative. This is not seen in P. vivax infections. There is often trophoblastic

Figure 43.12 Making a peripheral blood smear.

1

2

3

6

5

4

554 SECTION 9 Protozoan Infections

great extent on the experience of the microscopist, the quality of the slides, stains and microscope and the time spent examin-ing the slide. Artefacts are common and often confusing. Spe-ciation of malaria at the trophozoite stage is easier on the thin film, although gametocytes and schizonts are more likely to be seen on the thick film. The thin film is more accurate for para-site counting. The number of parasitized red cells per 1000 red cells should be counted. If there are two parasites in one red cell, this is counted as one. At low parasitaemias (<5/1000 on the thin film) the thick film should be counted; the number of parasites per 200, or preferably 500 white cells is noted. These figures can then be corrected by the total red cell and white cell counts to give the number of parasites per unit blood volume (µL). If the white count is not available then the count is assumed to be 8,000 µL. An alternative is to count all parasites in a fixed volume of blood. In severe malaria parasitaemias are usually high and the stage of parasite development should be assessed on the thin film. The proportion of asexual parasites containing visible pigment (i.e. mature trophozoites and schiz-onts) should be counted. The presence of pigment in neutro-phils and monocytes should also be noted and counted. In patients who have already received antimalarial treatment, pigment may still be present in leukocytes after clearance of parasitaemia and this is an important clue to the diagnosis. Monocytes containing pigment are cleared more slowly than pigment containing neutrophils. The morphological character-istics of human malaria parasites are given in Table 43.3.

ANTIGEN DETECTION METHODS

The introduction of simple, rapid, sensitive, highly specific and increasingly affordable dipstick or card tests for the diagnosis of malaria has been a major advance in recent years. These are based on antibody detection of malaria-specific antigens in blood samples; currently histidine-rich protein 2 (Pf HRP2), parasite lactate dehydrogenase (which is antigenically distinct from the host enzyme) and aldolase. Current Pf HRP2 and Pf LDH tests, based on colour reactions, provide a diagnostic sensitivity for P. falciparum similar to trained microscopists. Many tests also include ‘pan-malaria’ antibody which detects all malaria species or a specific P. vivax LDH or aldolase antibody.24 The cards or sticks then carry two band colour reactions plus a control (which should be positive). Thus a positive test with these two bands, with a negative in the Plasmodium falciparum test, signifies one of the other malaria species (or P. vivax for the specific tests) is causing the infection. This part of the test usually is less sensitive than good microscopy. The antigen detection tests may remain positive in patients with persistent gametocytaemia. There are now many different tests from many different manufacturers, based on several different antibodies and the considerable variability between them in performance characteristics has been reduced as a result of regular WHO evaluations (see: http://www.who.int/malaria/publications/atoz/9789241502566/en/index.html). Most current tests are based on Pf HRP2. These tests are the least expensive, simplest to perform and the most robust under tropical conditions. Vari-ability in the diagnostic sensitivity of Pf HRP2-based tests also results from sequence diversity in the gene encoding Pf HRP2 and consequent variability in the number of antigenic repeats. Because Pf HRP2 is cleared very slowly from the blood, these tests may remain positive for up to a month after the acute

needle. Two drops of blood are placed at one end of the slide. The thin film is made immediately by placing the smooth leading edge of a second (spreader) slide in the central drop of blood, adjusting the angle (less blood – more acute) and, while holding the edges of the slide, smearing the blood with a swift and steady sweep along the surface. If the blood drop is too large, the spreader slide should be dunked in the drop, then ‘jumped’ to the slide surface carrying a smaller amount of blood – and then smeared. Making good thin films requires some practice. Anaemic blood smears poorly. The thick film should be stirred in a circular motion with the corner of the second slide until clotting takes place. The thick film must be of uneven thickness, but it should be possible to read the hands, but not the figures, of a watch face through the film.

INTRADERMAL SMEARS

Chinese researchers have shown that smears from intradermal blood may contain more mature forms of P. falciparum than the peripheral blood. This is considered to allow a more com-plete assessment of severe malaria. The intradermal smears may also be positive or may show pigment containing leukocytes after the blood smear is negative. In terms of diagnostic sensi-tivity the intradermal smear is similar to the bone marrow (i.e. slightly more sensitive than peripheral blood). The smears are taken (Figure 43.13) from multiple intradermal punctures with a 25G needle on the volar surface of the upper forearm. The punctures should not ooze blood spontaneously, but sero-sanguinous fluid can be expressed on to the slide by squeezing.

STAINING AND READING

The thick film should be dried thoroughly otherwise it may wash away during staining. The thin film is then fixed in anhy-drous methanol (taking care not to fix the thick film). Giemsa’s stain buffered to a pH of 7.2 makes the best malaria slides, but for optimum results the stain should be left on the slide for 30 min. Field’s stain is quicker, but the thin and thick films are treated differently. The thin film is immersed in the red stain (Field’s B) for 6 seconds (s), then gently washed off for 5 s; then immersed in the blue stain (Field’s A) for 3–4 s and then gently washed off (5 s). The reverse order applies to the thick film: the slide is first immersed in the blue stain (Field’s A) for 5 seconds, then gently washed off (5 s) then the red stain (Field’s B) for 5 s, then gently washed off (5 s). Slides should be dried in a slide rack before examining under oil immersion at a magnification of ×1000.

Best results are obtained with fresh filtered stains and anhy-drous methanol for fixing the thin film. Use of repeatedly reused stain, full of precipitates and particles, with methanol left to absorb atmospheric water and a poorly maintained fungus-infected microscope (familiar to all of us who work in the tropics) makes accurate parasite counting a lot more difficult.

Before going to oil immersion on the microscope, the slide should be scanned briefly under low magnification to identify the best area for detailed examination. For the thin film, the tail of the film should be examined; for the thick film the area of optimum thickness and staining and least artefact is chosen. The thick film is approximately 30 times more sensitive than the thin film, although sensitivity and specificity depend to a

43  Malaria 555

treatment. Pf HRP2 is present in parasitized erythrocytes but it is also secreted into plasma and plasma concentrations (which can be assessed semi-quantitatively from the intensity of the colour reaction) are a valuable guide to the parasite biomass and thus severity. The Pf HRP2 test has proved useful in detect-ing mixed P. falciparum and P. vivax infections where the former was not evident microscopically. False-positive tests may occur

infection, particularly if the original parasitaemia was high. This is a disadvantage in areas where transmission is high and infections frequent, but is very useful in the diagnosis of severely ill patients who have received previous antimalarial treatment. Their parasitaemia may have cleared but the Pf HRP2 test will remain strongly positive. In contrast Pf LDH is cleared rapidly from blood and so the test becomes negative within days of

Figure 43.13 Making an intradermal smear.

DermisA B

1

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556 SECTION 9 Protozoan Infections

POSTMORTEM DIAGNOSIS

The diagnosis of cerebral malaria postmortem can be con-firmed from a brain smear. A needle aspirate or biopsy is obtained through the superior orbital foramen or the foramen magnum. A smear of grey matter is examined after staining the slide in the same way as for a thin blood film. Capillaries and venules are identified under low magnification microscopy and then examined under high magnification (×1000). If the patient died in the acute stage of cerebral malaria, the vessels are packed with erythrocytes containing mature parasites and a large amount of malaria pigment.

Clinical Course and ManagementThe clinical manifestations of malaria are dependent on the premorbid immune status of the host. In areas of intense P. falciparum malaria transmission, asymptomatic parasitaemia is usual in adults (premunition). Severe malaria never occurs in this age group: it is confined to the first years of life and becomes progressively less frequent with increasing age. In Africa before recent strengthening of control measures the average age of children admitted to hospital with severe malaria was 3 years and peak mortality was in the third year of life. The rate at which age-specific acquisition of premunition occurs is propor-tional to the intensity of malaria transmission. In areas with a constant high-level P. falciparum transmission (e.g. average infected anopheline biting frequencies of daily up to monthly), severe malaria occurs predominantly between 6 months and 3 years of age; milder symptoms are seen in older children and adults are usually asymptomatic and have low parasitaemias. Malaria is common in pregnancy, but is asymptomatic (although anaemia may be severe). The birth weight of babies born to

in patients with high concentrations of rheumatoid factor and false negatives have been reported in patients whose parasites have a rare antigenically variant form of Pf HRP2 (found occa-sionally in South America).

OTHER TECHNIQUES

Unlike mature red cells, malaria parasites contain DNA and RNA and malaria pigment. The nucleic acids can be stained with fluorescent dyes and visualized under ultraviolet light microscopy, or, with appropriate filters, seen under ordinary light and they can be amplified in PCR reactions. PCR is increas-ingly used in epidemiological assessments and is particularly useful in identifying parasite species in low-density infections. qPCR detection on 1 mL blood samples can reach detection thresholds 1000 times lower than the blood smear (10 parasites/mL). Quantitative assessment of gametocyte mRNA (e.g. QT NASBA) allows accurate quantitation of low-density gametocy-taemia. In the QBC™ technique blood samples are taken into a specialized capillary tube containing acridine orange stain and a float. Under high centrifugal forces (14 000 g) the infected erythrocytes, which have a higher buoyant density than unin-fected cells, become concentrated around the float. Using a modified lens adaptor (Paralens™) with its own light source, the acridine orange fluorescence from malaria parasites can be visualized through an ordinary microscope. Although slightly more sensitive than conventional light microscopy, it does not give parasite counts or speciation with accuracy and it is rela-tively expensive. It is useful for screening large numbers of blood samples rapidly. Detection of malaria antibody can be useful in some circumstances, such as confirmation of earlier infection and in epidemiological assessment of transmission intensity, but has no place in acute diagnosis.

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

Asexual parasites Usually only ring forms seen. Fine blue cytoplasm oval, circular, comma- shaped or occasionally thick band forms. squeezed to the edge of the cell (appliqué form). One or two chromatin dots. Parasitaemia may exceed 2%

Irregular large fairly thick rings becoming very pleomorphic as the parasite matures.

One chromatin dot

Regular dense ring enlarges to compact blue mature trophozoite.

One chromatin dot Low parasitaemia usual

Dense thick rings, maturing to dense round trophozoites Rectangular or band-form trophozoites Pigment associated with rings and trophozoites. Large red chromatin dot or band. Low parasitaemia usual

Meronts (Schizonts) Rare in peripheral blood.8–32 merozoites, dark brown-

black pigment

Common. 12–18 merozoites, orange brown pigment

8–14 merozoites, brown pigment

8–10 merozoites, black pigment

Gametocytes Banana-shaped.Male: light blue.Female: darker blue.Red-black nucleus with few

scattered blue-black pigment granules in cytoplasm

Round or oval.Male: round, pale

blue. Female: oval, dark blue. Triangular nucleus, few orange pigment granules

Large round dense blue like P. malariae, but prominent James’ dots.

Brown pigment

Large oval-shaped.Male: pale blue.Female: dense blue.Large black pigment granules

Red cell changes Normal size. As parasite matures cytoplasm becomes pale, the cells become crenated, and a few small red dots appear over the cytoplasm (Maurer’s clefts)

Enlarged. Pale red Schüffner’s dots increase in number as parasite matures

Cells become oval with tufted ends. Prominent James’ dots

Normal size and shape. No red dots

Note. Multiple invasion is often quoted as a feature of falciparum malaria. This is simply a function of higher parasite densities. At any given density multiple invasion is over three times more frequent with P. vivax compared with P. falciparum. P. knowlesi resembles P. falciparum in the first 8 hours of asexual development, then begins to resemble P. malariae when older.

TABLE 43.3 Morphological Characteristics of Human Malaria Parasites

43  Malaria 557

primigravidae is reduced significantly. Spleen rates are high (>50%) in children between 2 and 9, corresponding with the epidemiological terms hyperendemic and holoendemic malaria. Severe anaemia in young children is the most common presen-tation of severe falciparum malaria in these circumstances. With lower or more seasonal or unstable transmission patterns the age distribution of severe malaria shifts upwards, severe malaria is seen in older children as well and cerebral malaria becomes the most prominent manifestation. Spleen rates in children are lower than 50%. With even lower or more sporadic patterns of transmission and when non-immunes travel to endemic areas, symptomatic disease is seen at all ages. Although severe anaemia is a common presentation in children on the island of New Guinea where transmission of P. vivax is very high, other mani-festations of severe malaria do not occur commonly with P. vivax, P. ovale, or P. malariae. Nevertheless, the acute infection in a non-immune patient is still serious and debilitating. With more intense exposure a state of premunition is also reached with these infections.

INCUBATION PERIOD

In most cases of falciparum or vivax malaria the incubation period is approximately 2 weeks (Figures 43.14 and 43.15). Primary incubation periods can be long, particularly if the infection is suppressed by partially effective chemoprophylaxis. Most tropical strains of P. vivax had similar incubation periods to P. falciparum, but strains from cooler countries often had extremely long incubation periods. The primary infection began 8–12 months after sporozoite inoculation to coincide with the short summer-time mosquito breeding season in these cold countries. These strains of P. vivax (P. vivax var hibernans) acquired in northern and eastern Europe, Russia, central and

northern China, may now be extinct.3 The incubation period (time from sporozoite inoculation to fever) is prolonged by ineffective antimalarial treatment or prophylaxis – both of which reduce the effective multiplication rate. The durations of the prepatent and incubation periods are also strongly influ-enced by previous exposure, i.e. ‘immunity’. Effective immunity both reduces effective multiplication, which prolongs the pre-patent period and raises the threshold at which symptoms occur (premunition), which prolongs the incubation period. In vivax malaria the symptom threshold is raised disproportionately in immune individuals, i.e. the gap between the prepatent and incubation periods widens.

MIXED SPECIES INFECTIONS

The incidence of mixed species infections is always underesti-mated. Even with sensitive PCR detection methods, which reveal a much higher rate than microscopy, mixed infections are underestimated. In simultaneous infections with P. falci-parum and P. vivax, the former suppresses the latter and the primary vivax malaria infection may not appear until several weeks later. Sometimes the reverse occurs and P. vivax sup-presses P. falciparum. In sub-Saharan Africa P. falciparum commonly occurs together with P. malariae or P. ovale. In many areas outside Africa P. falciparum and P. vivax are both common and co-existent infections are frequent but, because of mutual suppression, the incidence is considerably underesti-mated. In Thailand, approximately 30% of patients with P. fal-ciparum malaria will have a subsequent symptomatic infection with P. vivax within 2 months of their primary falciparum malaria, without further exposure to malaria infection.25 In Myanmar, the figure is 50%. The converse (P. vivax malaria with undiagnosed coincident P. falciparum infection) occurs in

Figure 43.14 Asexual life cycle of Plasmodium falciparum with approximate ages of development. After some 13–16 hours of development the parasitized erythrocytes start to adhere to the vascular endothelium lining the capillaries and venules.

558 SECTION 9 Protozoan Infections

Figure 43.15 Asexual life cycle of Plasmodium vivax with approximate ages of development.

approximately 8% of cases. In a low transmission setting coin-cident infection of P. falciparum with P. vivax reduces the risk of severe malaria fourfold,26 reduces the degree of anaemia, and reduces P. falciparum gametocyte carriage. Mixed infections with P. malariae and P. ovale are also underestimated.

PYROGENIC DENSITY

The parasitaemia at which fever (>37.3°C) occurs is termed the ‘pyrogenic density’. This varies widely: some non-immune patients will become febrile before parasites are visible on blood smears (i.e. the incubation period is shorter than the prepatent period), whereas immune adults can sometimes tolerate up to >10,000 P. falciparum parasites/µL without fever. The pyrogenic density for P. vivax is generally lower than that of P. falciparum; in 76% of cases reported by Kitchen, the pyrogenic density was <100 parasites/µL. In P. falciparum infection average pyrogenic densities in non-immunes can be as high as 10 000/µL, but it must be remembered that less than half the life cycle circulates in falciparum malaria. The parasites in the blood smear are the circulating parasites in the generation subsequent to that which underwent pyrogenic merogony and they are therefore an underestimate of the total parasite burden. The pyrogenic density is a marker of immunity. High pyrogenic densities indi-cate premunition and a lower risk of severe disease. There are

less data on pyrogenic densities in P. malariae infections, but it appears that they are higher than for P. vivax; values over 500/µL were found in 38% of Boyd’s cases. There are limited data on P. ovale, but the available evidence suggests a pyrogenic density similar to P. vivax.

UNCOMPLICATED MALARIA

The clinical features of uncomplicated malaria are common to all five species although P. vivax, which tends to synchronize rapidly, may cause more severe symptoms early in the course of the infection and P. knowlesi infections with their 24-hour (quotidian) asexual cycle can rapidly develop into severe malaria. P. malariae and possibly P. ovale both have a more gradual onset than P. vivax. P. falciparum is unpredictable: the onset ranges from gradual to fulminant. The first symptoms of malaria are nonspecific and resemble influenza. Headache, muscular ache, vague abdominal discomfort, lethargy, lassitude and dysphoria often precede fever by up to 2 days. The tem-perature rises erratically at first, with shivering, mild chills, worsening headache and malaise, loss of appetite and some-times abdominal discomfort. Children are irritable, lethargic and anorexic. If the infection is left untreated the fever in P. vivax and P. ovale regularizes to a 2-day cycle (tertian) and P. malariae fever spikes occur every 3 days (quartan pattern).

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P. falciparum remains erratic for longer and may never regular-ize to a tertian pattern. These terms derive from the Greek practice of ‘inclusive reckoning’, in which the beginning of the fever is considered day 1. Thus, a tertian fever recurs every 3rd day and a quartan fever every 4th day, with intervals of 2 and 3 days, respectively. Some infections consist of two broods cycling 24 hours out of phase and in these there is a daily fever spike (quotidian fever). Even more complex fever patterns are described in detail in the early literature.

The classical malaria fever charts (which graced earlier edi-tions of this textbook) and the teeth-chattering rigors and profuse sweats that characterized the ‘paroxysm’ (Figure 43.16), are relatively unusual today as malaria therapy of neurosyphilis has been long abandoned (penicillin is more effective and more pleasant) and symptomatic infections are treated as soon as they are diagnosed. In a true paroxysm, the temperature usually rises steeply from a normal or slightly elevated level to exceed 39°C. As the temperature begins to rise there is intense head-ache and muscular discomfort. The patient feels cold, clutches at blankets and curls up shivering and uncommunicative (the chill). There is peripheral vasoconstriction and often ‘goose-pimples’. Within minutes, the limbs begin to shake and the teeth chatter and the temperature climbs rapidly to a peak (usually between 39°C and 41.5°C). The rigor usually lasts 10–30 min, but can last up to 90 min (Figure 43.16). By the end of the rigor, there is peripheral vasodilatation and the skin feels hot. A profuse sweat then breaks out. The blood pressure is relatively low and there may be symptomatic orthostatic hypotension. The patient feels exhausted and may sleep. Defervescence usually takes 4–8 hours. Paroxysms with rigors are more common in P. vivax and P. ovale than in P. falciparum or P. malariae malaria. They may herald a relapse, or occur after several days of more chaotic fever in primary infections with these two malaria species. True rigors are unusual in naturally acquired falciparum malaria. As the infection continues the spleen and liver enlarge and anaemia develops and the patient loses weight. If no treatment is given the natural infection sta-bilizes for several weeks or months and then gradually resolves.

Figure 43.16 The rigor of P. vixax malaria. True rigors are rare in P. falciparum malaria. In infections with the other three human malaria parasites rigors occur when the parasite population has synchronized sufficiently. (The time course of signs and symptoms is taken from Kitchen & Puttnam: J Natl Malaria Soc 1946;5:57–78.)

Hours

0 1 2 3 4 5 6 7 8 9

Tem

pera

ture

(ºC

)

‘Cold’ stage

51 48 451 Minutes

Diaphoresis

Peak temperature in 65% casesoccurs from 1500 to 2000 hours

Mean parasite density at rigor = 4.012 (669)/µL Mean pyrogenic density = 274 (140)/µL

Plasmodium vivax The rigor

Rigors

Impendingsensations

‘Hot’stage

37

38

39

40

41

The duration of illness is proportional to the level of immunity and differs between the parasite species. Mild abdominal dis-comfort is common in malaria and rarely patients may appear to have an ‘acute abdomen’. Either constipation or diarrhoea may occur. In some areas, watery diarrhoea is a prominent manifestation. However, there is usually no difficulty distin-guishing malaria from gastroenteritis. A dry cough is relatively common but is not prominent. However, the respiratory rate may be raised, particularly in children and this can give rise to diagnostic confusion in primary healthcare facilities where respiratory rate is used as the only criterion for the diagnosis of acute respiratory infection. On chest examination there is no evidence of consolidation or effusion, but in an endemic area the clinical distinction between early pneumonia and severe malaria in young children can be very difficult. In routine clini-cal practice in malarious areas of the tropics, malaria is the most common cause of fever in children and is the most likely diag-nosis in a febrile patient with no obvious respiratory or abdom-inal abnormalities. In travellers returning from such areas, any fever must be considered to be malaria unless proved otherwise. In semi-immune patients low-grade fever may be the only com-plaint in malaria. In tropical practice, malaria is so common that it must be excluded in any febrile patient.

RELAPSE

Both P. vivax and P. ovale have a tendency to relapse after resolu-tion of the primary infection. Relapse, which results from matu-ration of persistent hypnozoites in the liver, must be distinguished from recrudescence of the primary infection because of incom-plete treatment. P. falciparum is the usual cause of recrudescent infections and these tend to arise 2–4 weeks following treatment (but this can be as long as 10 weeks following treatment with slowly eliminated drugs). Relapses occur weeks or months (or even years) after the primary infection. The proportion of cases relapsing and the intervals between relapses vary between strains and depends on the size of the inoculum and the inten-sity or previous exposure. The pattern of relapse is also deter-mined by the geographic origin of the infection. For example, over 50% of P. vivax infections in Thailand relapse whereas in most of India the proportion is closer to 20%.3 The subtropical P. vivax has an interval usually of 8–10 months between primary illness and relapse whereas tropical strains have frequent relapses at short intervals (3–6 weeks depending on the drugs used). In Patrick Manson’s famous experiment, conducted in September 1900, he infected his 23-year-old son with P. vivax, through mosquitoes sent by rail from Rome to London. His son became ill with ‘double tertian fever’, but was treated with quinine and recovered fully. In June 1901 (i.e. 9 months later), he suddenly became ill again with vivax malaria; a relapse inter-val of 9 months. In recent years, a relapse interval of 6 weeks has been quoted widely for tropical Plasmodium vivax but this is an artefact of the use of chloroquine which suppresses the first relapse (which would otherwise emerge at 3 weeks). The symptoms of a relapse start more abruptly than in the primary infection as the infection is more synchronous. They may begin with a sudden chill or rigor.

MALARIA IN PREGNANCY

Malaria (all species) in early pregnancy causes abortion. In areas of intense transmission the principal impact of falciparum

560 SECTION 9 Protozoan Infections

malaria in pregnancy is an increased incidence of anaemia and a reduction in birth weight (approx. 170 g on average) of babies born to primigravidae. Thus, a greater proportion of babies have low birth weights (<2.5 kg). Low birth weight is a major risk factor for infant death. Malaria reduces birth weight mainly by intrauterine growth retardation (IUGR). In high transmis-sion areas, malaria may also cause prematurity. In low transmis-sion areas, prematurity is caused by symptomatic malaria close to term, not earlier in the pregnancy. The net result is an increased risk of neonatal death. In high transmission areas despite intense sequestration of P. falciparum parasites in the placenta, the mothers are usually asymptomatic, although they are more likely to be anaemic. In areas with lower levels of malaria transmission (mesoendemic or hypoendemic) symp-tomatic disease occurs and pregnant women are at an increased risk of severe falciparum malaria, particularly in the 2nd and 3rd trimesters. In low transmission areas, the adverse effects of malaria on birth weight extend to the first three pregnancies (and in non-immunes, to all pregnancies). Again anaemia is common and there is an increased risk of developing severe malaria. Anaemia itself is a risk factor for maternal mortality; moderate anaemia (Hb 4–8 g/dL) carrying a relative risk of 1.35 and severe anaemia (Hb <4 g/dL) a risk of 3.5. If a pregnant woman does develop severe malaria, fetal loss is common and the maternal mortality is very high. The mortality of cerebral malaria in pregnancy is approximately 50%, compared with 15–20% in non-pregnant adults. Acute pulmonary oedema and hypoglycaemia are particular complications of severe malaria in pregnancy. The baby is commonly stillborn. The clinical features of uncomplicated vivax, ovale or malariae malaria are similar to those of uncomplicated P. falciparum. P. vivax infec-tions also increase anaemia and they reduce birth weight by approximately 100 g. In contrast to P. falciparum, vivax malaria affects multigravidae more than primigravidae. If a mother delivers with acute malaria bloodborne transmission to the newborn is not uncommon, but this often resolves spontane-ously. Nevertheless, babies must be observed closely for con-genital malaria and malaria considered in the differential diagnosis of neonatal fever or anaemia.

MALARIA IN CHILDREN

The majority of childhood malaria infections (Figure 43.17) present with fever and malaise and respond rapidly to antima-larial treatment. Severe falciparum malaria is rare in infancy, although when it does occur the mortality is high. In young children the progression of falciparum malaria can be rapid. Generalized seizures are associated with fever, but they are more common in P. falciparum than P. vivax malaria, even in the absence of other signs of cerebral involvement. This suggests that cerebral sequestration causes significant pathology even in conscious patients. Coma, convulsions, acidosis, hypoglycaemia and severe anaemia are common presenting features of severe malaria in childhood. At the bedside, the presence of respiratory distress (acidotic breathing) or deep coma defines children at high risk of dying. These two clinical syndromes account for the majority of lethal infections. In areas of intense transmis-sion profound anaemia is the usual manifestation of severe malaria and this occurs mainly in the 1–3-year age group. Severe malaria is rare in older children in high transmission settings. In areas of lower, less stable transmission cerebral malaria becomes a predominant manifestation of severe disease and the

Figure 43.17 A 6-year-old Thai boy with cerebral malaria. His father was admitted at the same time with cerebral malaria – both survived. (B) A 3-year-old Gambian girl with cerebral malaria and opisthotonos. (Courtesy of Jane Crawley.)

A

B

age range shifts upwards. Jaundice and pulmonary oedema are unusual in young children and renal failure requiring dialysis or haemofiltration is very rare (a significant difference com-pared with adults). As a consequence iatrogenic overhydration is less of a problem than in adults, although intravenous fluid administration must still be carefully supervised in small chil-dren. Dehydration is more common but rapid fluid loading is potentially lethal. In cerebral malaria seizures occurs frequently, particularly in the <3-year age group and should be treated promptly. Hypoglycaemia is common, occurring in up to 30% of children with severe malaria and is often accompanied by lactic acidosis. The blood glucose should be checked frequently and, where possible, continuous intravenous infusions of 5% or 10% dextrose given as a preventive measure.

In general children tolerate the antimalarial drugs better than adults and their symptoms resolve more quickly. The temptation to estimate body weight by ‘eye’ should be resisted and all children should be weighed if possible so that the doses of antimalarial drugs can be given on a mg/kg basis. Although administration of drugs adjusted to surface area is theoretically preferable, antimalarial doses have been devised on the basis of body weight. Children with acute malaria vomit readily, particularly if the temperature is high. Oral antimalarial

43  Malaria 561

fulminant disease with the rapid development of anaemia, aci-dosis, pulmonary oedema and acute kidney injury as the major manifestations. Coma does not occur.

Falciparum malaria is the major cause of death from malaria. The progression to severe disease can be rapid. In young chil-dren presenting with cerebral malaria a history of less than 1 day’s illness is common. Although undernutrition is associ-ated with an increased risk of clinical malaria and anaemia in high transmission settings, cerebral malaria is rare in severe malnutrition and often seems to strike down the healthiest people. The great malariologist, Ettore Marchiafava, noted over 100 years ago how common severe malaria was in the ‘hale and hearty’ Italian shepherds who descended from the malaria-free mountains to the malarious valleys every autumn. In adults, patients with severe malaria usually have a history of being ill for several days before admission to hospital.

Definitions of severe falciparum malaria are useful for clini-cal and epidemiological purposes. Definitions were proposed by working groups convened by the World Health Organization (WHO) in 1986, 1990 and 2000 and they are currently being revised.27 In severe malaria, there is often evidence of multiple vital organ dysfunction and more than one of the above criteria are fulfilled (Table 43.4A,B). Strictly defined severe malaria has a mortality of approximately 10% in children and 15% in adults but this depends on the degree of vital organ dysfunction. Of the various major criteria severe anaemia (Hb<5 g/dL) carries a much better prognosis than evidence of severe cerebral, renal or metabolic dysfunction. Physicians should not worry unduly about definitions or semantics. They should treat any patient about whom they are worried as having severe malaria, even if they do not fall clearly into one of the above categories.

CEREBRAL MALARIA

This may be defined strictly as unrousable coma (i.e. there is a non-purposeful response or no response to a painful stimulus) in falciparum malaria. This is usually a Glasgow Coma Score of <11 or in young children a Blantyre Coma Score of <3. In prac-tice, any patient with altered consciousness should be treated for severe malaria. Although cerebral malaria is the most prom-inent feature of severe falciparum malaria, some patients with ultimately lethal infections never lose consciousness until they die. In cerebral malaria the onset of coma may be sudden, often following a generalized seizure, or gradual, with initial drowsi-ness, confusion, disorientation, delirium or agitation, followed by unconsciousness. Extreme agitation is a poor prognostic sign in falciparum malaria. The length of the prodromal history is usually several days in adults, but in children can be as short as 6–12 hours. A history of convulsions is common.

On examination the patient is febrile and unrousable. There may be some passive resistance to head flexion, but the board-like rigidity of meningitis is not found and there are no other signs of meningeal irritation. There may be anaemia, which in some cases, particularly children, may be profound. Conversely jaundice is relatively unusual in children but common in adults. Signs of bleeding are unusual and indicate a poor prognosis. The patient is usually warm, dry and well perfused peripherally, with a low-normal blood pressure and a sinus tachycardia. Skin perfusion is variable. Poor capillary refill (refill time >2 s) is a serious prognostic sign in children. Intermittent ‘goose-pimples’ are common in association with cutaneous vasoconstriction. Sustained hyperventilation is a poor prognostic sign as it

treatment is more likely to be retained if it is palatable and the child is cool and calm before drug administration. In busy tropical clinics, only a minority of patients can be admitted to hospital and many children with moderately severe malaria have to be treated on an outpatient basis. It was common prac-tice to administer a single dose of parenteral quinine and to send the patient home with the remainder of the oral regimen and to give the parents advice to return if the child deteriorated further. In this situation there is a danger of significant iatro-genic hypotension if the child is kept upright (e.g. on the moth-er’s back). If possible, the child should be observed for at least 2 hours following parenteral drug administration and reas-sessed before discharge.

The diagnosis of severe malaria in children living in malaria-endemic areas may be difficult. As a positive blood smear is common in apparently healthy children, finding malaria para-sites in the blood of a sick child does not necessarily mean the child has severe malaria. Fever and rapid laboured breathing could be pneumonia even if the blood smear is positive. The obtunded child might have meningoencephalitis and the shocked child might be septicaemic despite positive blood smears. The net result is that severe malaria in children tends to be overdiagnosed.

MALARIA AND HIV

When the enormity of the HIV epidemic in Africa was first recognized it was thought that malaria and HIV infection did not interact significantly. This is not true. While asymptomatic HIV infection has little impact on malaria, with increasing immunosuppression in HIV-AIDS immune control of malaria is impaired. There is an increasing risk of parasitaemia, increas-ing risk of illness, and in low transmission settings an increased risk of severe malaria. HIV infection compounds malaria- associated reduction in birth weight. Therapeutic responses to antimalarial treatment are impaired so treatment failure rates are increased and preventive therapies less effective. Drug inter-actions between antiretrovirals and antimalarials have not been studied in sufficient detail yet. Where sulfadoxine-pyrimethamine is effective, then prophylaxis against opportu-nistic infections with trimethoprim-sulfamethoxazole will protect from malaria also.

SEVERE MALARIA

Death from acute P. vivax, P. ovale or P. malariae infections is very rare. Occasionally, already debilitated patients, or those with another disease process may succumb and fatal haemor-rhage may follow a ruptured spleen (either traumatic or spon-taneous), but these events are uncommon. Severe anaemia may follow repeated P. vivax infections and is an important present-ing feature in children in high transmission settings. Pulmonary oedema carries a better prognosis in vivax than in falciparum malaria but can be lethal. There have been many case reports of ‘cerebral vivax malaria’. Some of these may have been misdi-agnoses, but there are several recent reports of severe Plasmo-dium vivax infections from Indonesia, India and South America. In low transmission settings the case-specific risk of developing severe malaria with vivax malaria is substantially lower (>100 fold) than with falciparum malaria and even in high transmission settings it is over ten times lower. The simian parasite P. knowlesi is potentially lethal, capable of producing

562 SECTION 9 Protozoan Infections

1. Cerebral malaria – unrousable coma not attributable to any other cause in a patient with falciparum malaria. The coma should persist for at least 30 min (1 hour in the 2000 definition) after a generalized convulsion to make the distinction from transient postictal coma. Coma should be assessed using the Blantyre Coma Scale in children or the Glasgow Coma Scale in adults (see Table 43.14).

2. Severe anaemia – normocytic anaemia with haematocrit <15% or haemoglobin <5 g/dL in the presence of parasitaemia more than 10 000/µL. Note that finger prick samples may underestimate the haemoglobin concentration by up to 1 g/dL if the finger is squeezed. If anaemia is hypochromic and/or microcytic, iron deficiency and thalassaemia/haemoglobinopathy must be excluded. (These criteria are rather generous; and would include many children in high transmission areas. A parasitaemia of >100 000/µL might be a more appropriate threshold).

3. Renal failure – defined as a urine output of <400 mL in 24 hours in adults, or 12 mL/kg in 24 hours in children, failing to improve after rehydration, and a serum creatinine of more than 265 µmol/L (>3.0 mg/dL). (In practice for initial assessment, the serum creatinine alone is used).

4. Pulmonary oedema or adult respiratory distress syndrome.5. Hypoglycaemia – defined as a whole blood glucose concentration of <2.2.mmol/L (40 mg/dL).6. Circulatory collapse or shock – hypotension (systolic blood pressure <50 mmHg in children aged 1–5 years or <70 mmHg in adults), with

cold clammy skin or core-skin temperature difference >10°C. (The more recent review declined to give precise definitions, but noted the lack of sensitivity or specificity of core-peripheral measurements.) Capillary refill time is not mentioned but recent studies indicate this simple test provides a good assessment of severity.

7. Spontaneous bleeding from gums, nose, gastrointestinal tract, etc. and/or substantial laboratory evidence of DIC. (This is relatively unusual.)8. Repeated generalized convulsions – more than two observed within 24 hours, despite cooling. (In young children, these may be febrile

convulsions, and the other clinical and parasitological features need to be taken into account.) Clinical evidence of seizure activity may be subtle (e.g. tonic clonic eye movements, profuse salivation, delayed coma recovery).

9. Acidaemia – defined as an arterial or capillary pH<7.35 (note temperature corrections are needed as most patients are hotter than 37°C; add 0.0147 pH unit per degree Celsius (°C) over 37°C), or acidosis defined as a plasma bicarbonate concentration <15 mmol/L or a base excess >10. (Operationally the clinical presentation of ‘respiratory distress’ or ‘acidotic breathing’ is focussed upon in the 2000 recommendations. Abnormal breathing patterns are a sign of severity indicating severe acidosis, pulmonary oedema or pneumonia).

10. Macroscopic haemoglobinuria – if definitely associated with acute malaria infection and not the result of oxidant antimalarial drugs in patients with erythrocyte enzyme defects such as G6PD deficiency. (This is difficult to ascertain in practice: if the G6PD status is checked following massive haemolysis, the value in the remaining red cells may be normal even in mild G6PD deficiency. This part of the definition is not very useful.)

11. Postmortem confirmation of diagnosis. In fatal cases a diagnosis of severe falciparum malaria can be confirmed by histological examination of a postmortem needle necroscopy of the brain. The characteristic features, found especially in cerebral grey matter, are venules/capillaries packed with erythrocytes containing mature trophozoites and schizonts of P. falciparum. (These features may not be present in patients who die several days after the start of treatment, although there is usually some residual pigment in the cerebral vessels.)

The 2000 recommendations also included the following (Table 43.4B)

12. Impairment of consciousness less marked than unrousable coma. (Any impairment of consciousness must be treated seriously assessment using the Glasgow Coma Scale is straightforward, but the Blantyre Scale needs careful local standardization particularly in younger children.)

13. Prostration: Inability to sit unassisted in a child who is normally able to do so. In a child not old enough to sit, this is defined as an inability to feed. This definition is based on examination not history. Prostration alone, without other signs of severity, carries a relatively low mortality.

14. Hyperparasitaemia – the relation of parasitaemia to severity of illness is different in different populations and age groups, but in general very high parasite densities are associated with increased risk of severe disease, e.g. >4% parasitaemia is dangerous in non-immunes, but may be well tolerated in semi-immune children. In non-immune children studied in Thailand a parasitaemia ≥4% carried a 3% mortality (30 times higher than in all uncomplicated malaria) but in areas of high transmission values much higher may be tolerated well. Whatever the circumstances a parasitaemia ≥20% indicates severe malaria.

The following were not considered criteria of severe malaria: Jaundice – detected clinically or defined by a serum bilirubin concentration >50 µmol/L (3.0 mg/dL). This is only a marker of severe malaria

when combined with evidence of other vital organ dysfunction such as coma or renal failure). Hyperpyrexia – a rectal temperature above 40°C in adults and children is no longer considered a sign of severity.

TABLE 43.4A 1990 WHO Definition of Severe Malaria

GROUP 1Children at immediately increased risk of dying who require parenteral antimalarial drugs and supportive therapyProstrated children (prostration is the inability to sit upright in a child normally able to do so, or to drink in the case of children too young to sit) Prostrate but fully conscious Prostrate with impaired consciousness but not in deep coma Coma (the inability to localize a painful stimulus)Respiratory distress (acidotic breathing) Mild – sustained nasal flaring and/or mild intercostal indrawing (recession) Severe – the presence of either marked indrawing (recession) of the bony structure of the lower chest wall or deep (acidotic) breathing

GROUP 2Children who, though able to be treated with oral antimalarial drugs, require supervised management because of the risk of clinical

deterioration, but who show none of the features of group 1 (above)Children with a haemoglobin level <5 g/dL or a haematocrit <15%Children with two or more convulsions within a 24-h period

GROUP 3Children who require parenteral treatment because of persistent vomiting but who lack any specific clinical or laboratory features of groups 1

or 2 (above).

TABLE 43.4B Outline Classification of Severe Malaria in Children (WHO 2000)

43  Malaria 563

the mortality. In the Vietnam War, the mortality from acute falciparum malaria was higher in soldiers who had returned to the USA than it was in Vietnam. Obviously the diagnosis was made much more rapidly in Vietnam where physicians were well aware of malaria, than in the USA, where they were not.

CONVULSIONS

Seizures are common, particularly in young children. They are associated with falciparum malaria even in uncomplicated infections. In the majority of cases the child recovers unevent-fully following one or two generalized convulsions, but some patients do not recover consciousness rapidly (<30 min) and may remain unrousable (cerebral malaria). In some cases the cause of the protracted coma is status epilepticus. Focal seizures may also occur, but they are less common. Aspiration pneumo-nia is a common and preventable sequel to grand mal seizures. Repeated grand mal seizures in cerebral malaria are associated with residual neurological sequelae.

POST-MALARIA NEUROLOGICAL SYNDROMES AND DEFICITS

In approximately 1–3% of adults and 10–23% of children there is a clinically obvious persistent neurological deficit following cerebral malaria (Figure 43.18). In children, this is associated with preceding profound and protracted coma, anaemia and prolonged and repeated convulsions. In a retrospective study from Kenya, multiple seizures were associated with persistent motor deficits, malnutrition, hypoglycaemia and seizures with subsequent language deficits and deep coma with cognitive impairment. In The Gambia hypoglycaemia was not a risk factor for neurological deficit. About 10% of children have demonstrable language deficit following cerebral malaria. There is also an increased risk of epilepsy following severe malaria in childhood. As severe malaria and seizures associated with malaria are so common in children, subtle but significant psy-chomotor impairment is of tremendous importance to tropical countries.28 It is often difficult to distinguish a pre-existing neu-rological condition ‘revealed’ by symptomatic malaria from a malaria-induced condition, but it is becoming increasingly clear

indicates metabolic acidosis if the chest is clear on clinical examination, or pneumonia or pulmonary oedema if it is not. The liver and spleen are commonly enlarged, but soft. Massive splenomegaly is not found. There is no lymphadenopathy and no rash. The clinical features are usually of a symmetrical encephalopathy. Focal signs are unusual. On examination of the nervous system the gaze is usually normal or divergent (but there is no evidence of extraocular muscle paresis) (Figure 43.13). The pupils are usually mid-size and equally reactive. The fundus should be examined carefully. Five distinct funduscopic abnormalities have been observed; retinal whitening, retinal haemorrhages, focal whitening of vessels, papilloedema and cotton wool spots.17 This retinopathy is highly specific for fal-ciparum malaria and is more easily seen using indirect ophthal-moscopy. Papilloedema is unusual and is a sign of poor prognosis, as is retinal oedema. Retinal haemorrhages are common. The haemorrhages, which rarely affect the macula, are often flame- or boat-shaped and may have a pale centre resembling Roth spots. The retinal vessels should be examined for a very characteristic segmental whitening that probably reflects intense sequestration with red cells containing little hae-moglobin and mature parasites. High-resolution digital imaging retinal angiography shows irregular vascular lining in some vessels and obstruction reflecting cytoadherence.17 In adult patients, the corneal reflexes are usually preserved but in chil-dren with deep coma they may be lost (a poor prognostic sign). It is important to examine the eyes carefully to exclude the rapid repetitive jerky movements that indicate seizure activity. There may be forced jaw closure with repetitive spontaneous teeth grinding (bruxism). The jaw jerk is sometimes brisk and there is often a pout reflex. Other frontal release signs are very unusual. Cranial nerve abnormalities are rare. Tone may be increased, decreased or normal. Likewise the reflexes can be brisk or depressed. The abdominal reflexes are invariably absent, the cremasteric reflexes often preserved and the plantar responses extensor in approximately half the patients. Patients may exhibit phasic increases in tone with extensor posturing of the decorticate (arm flexed, legs extended), or more usually, decerebrate (arms and legs extended) types. The back may arch as in opisthotonos, with sustained, usually upward and lateral, ocular deviation. The posturing is commonly associated with noisy hyperventilation. Generalized or sometimes focal seizures may occur. The duration of coma varies considerably but overall is shorter in children (average 1 day) than in adults (average 2–3 days). Clinical evidence for seizure activity may be very subtle (e.g. tonic clonic eye movements without limb move-ment) and in some children there are no signs despite electro-encephalographic evidence. Aspiration pneumonia is a potentially lethal sequel.

Untreated cerebral malaria is probably nearly always fatal. The overall mortality of treated cerebral malaria obviously depends on the referral practices and medical facilities available, but in reported studies with quinine treatment averaged 15% in children and 20% in adults (but up to 50% in pregnancy). Some series reported lower mortalities, but in these the defini-tion of cerebral malaria has been more ‘generous’, i.e. they have included patients who were prostrated, obtunded or delirious but not unrousable. Treatment with artesunate reduces this mortality by between one-fifth (children) and one-third (adults). Hospitals acting as secondary or tertiary referral centres often experience higher mortalities as they see a residue of more severe patients. The later the patient is referred, the higher

Figure 43.18 Permanent global residual neurological deficit following prolonged hypoglycaemia in a 33-year-old Vietnamese woman who had cerebral malaria in pregnancy. She had received intravenous quinine but despite parenteral glucose administration became repeatedly hypoglycaemic.

564 SECTION 9 Protozoan Infections

METABOLIC ACIDOSIS

The main clinical indication of metabolic acidosis is laboured hyperventilation with increased inspiratory effort (often termed respiratory distress) and a clear chest on auscultation (Kussmaul’s breathing). This usually results from accumulation of organic acids including lactic acid, but ketoacidosis may be present in children. There is a wide anion gap. Hypovolaemia must be corrected but recent evidence suggests that this does not play a major role in causing acidosis. In areas where aspirin is still used widely salicylate intoxication should be considered. Acidosis may be associated with renal failure in adults, but in the acute infection there is also a lactic acidosis. There may be a temporary worsening of lactic acidosis following grand-mal seizures, but the outlook for persistent acidosis is poor. Although blood pressure and tissue perfusion are usually adequate ini-tially, hypotension commonly ensues.

BLACKWATER FEVER

The sinister reputation of blackwater fever derives from the high mortality (20–30%) documented in Europeans and Asians working in colonial Africa in the first half of the twentieth century. However the passage of black or dark-brown-red urine (blackwater) is often not associated with significant renal impairment. Blackwater is usually transient and resolves without complications, but in severe cases AKI may develop. This behaves as acute tubular necrosis. Blackwater results from massive hae-molysis. In some patients myoglobinuria may also be present. Transfused blood is also rapidly haemolysed. The mortality is highest when blackwater fever is associated with severe malaria and other evidence of vital organ dysfunction. Patients with blackwater fever and severe anaemia often have a slate-grey appearance and their plasma may be red (haemoglobinaemia).

ACUTE PULMONARY OEDEMA

Hyperventilation or Kussmaul’s breathing (sometimes termed respiratory distress) is a poor prognostic sign in malaria. In the tachypnoea associated with high fever, breathing is shallow compared with the ominous laboured hyperventilation associ-ated with metabolic acidosis, pulmonary oedema or broncho-pneumonia. Acute pulmonary oedema (acute respiratory distress syndrome) may develop at any time in severe falci-parum malaria. It is particularly common in pregnant women, but rare in children. In some cases malaria ARDS may be dif-ficult to distinguish clinically from pneumonia. The heart sounds are normal. The central venous pressure and pulmonary artery occlusion pressures are usually normal, the cardiac index is high and systemic vascular resistance is low. This points to an increase in capillary permeability (unless the patient has been overhydrated). The chest radiograph shows increased intersti-tial shadowing and a normal heart size.

HYPOTENSION

The majority of patients with severe malaria are febrile, with a high cardiac output, a low systemic vascular resistance and a low-normal blood pressure. They are usually warm and well perfused. Patients with severe disease may develop sudden hypotension and become shocked. This was called ‘algid malaria’.

that subtle but important neurocognitive deficits may follow recovery from cerebral malaria, particularly in children. The long-term prognosis of these has not yet been established.

In approximately 60% of severe cases with residual neuro-logical deficit, there is a hemiparesis with variable hemisensory deficit and sometimes hemianopia. Cortical blindness, diffuse cortical damage, tremor and occasionally isolated cranial nerve palsies may occur. Many of these substantial deficits recover rapidly and by 6 months only 4% of survivors have clinically obvious neurological abnormalities.

Rarely patients who recover from cerebral malaria may lapse into coma again, usually after a period of 1–2 days when they are rousable. In this condition the CSF protein may be elevated (200–300 mg/dL) and there is sometimes an increase in CSF lymphocytes. There may be residual neuro-logical deficit on recovery. A variety of other late neurological complications may occur following recovery from cerebral malaria. These include psychosis, encephalopathy, Parkinsonian rigidity and tremor, a fine tremor and cerebellar dysfunction. These post-malaria neurological syndromes (PMNS) may also rarely follow uncomplicated malaria and could account for some of the cases previously attributed to mefloquine or chlo-roquine neurotoxicity. On the other hand there appears to be a strong interaction between mefloquine and cerebral malaria such that 5% of patients who receive mefloquine after severe malaria develop PMNS (a risk 10–50 times higher than follow-ing mefloquine treatment of uncomplicated malaria). Meflo-quine should therefore not be used following cerebral malaria. The conditions are self-limiting, but very distressing and usually resolve over several days, or sometimes 1–2 weeks. The syn-drome of cerebellar ataxia occurring 2–3 weeks after acute uncomplicated malaria appears to be relatively common in Sri Lanka. It too is usually self-limiting with recovery over a few weeks.

ACUTE KIDNEY INJURY

In some adult patients with severe malaria, acute oliguric renal impairment and other vital organ dysfunction is present on admission, whereas in others renal dysfunction becomes evident as the patient recovers from the acute phase of severe disease. In the fulminant presentation, there is a high incidence of asso-ciated hepatic dysfunction and metabolic acidosis and pulmo-nary oedema is the usual terminal event. The blood pressure is normal. Jaundice is common and there may be a bleeding ten-dency. There may be slight proteinuria, but the urine sediment is unremarkable. The subacute presentation carries a better prognosis. The patient may be oliguric but is rarely anuric. The serum creatinine rises over a period of days until either dialysis is required because of hyperkalaemia or uraemic complications such as bleeding, pleural or pericardial effusions, encephalopa-thy or intractable vomiting, or there is gradual resolution with an increase in urine output. In the subacute presentation of acute renal impairment parasitaemia may have cleared follow-ing antimalarial treatment before the patient is referred to hos-pital. Although AKI is a common complication of malaria in adults living in areas of low or unstable transmission and ele-vated blood urea is an important manifestation of severe malaria in children, it is very unusual for children to require renal replacement therapies. AKI is also associated with haemo-globinuria in patients with massive haemolysis (see Blackwater Fever, below).

43  Malaria 565

Laboratory FindingsThere is a progressive normochromic normocytic anaemia. The white count is usually normal, but may be raised in very severe malaria and very occasionally there is a leucoerythroblastic picture. There is slight monocytosis, lymphopenia and eosino-penia, with reactive lymphocytosis and eosinophilia in the weeks following the acute infection. The platelet count is reduced in all acute malarias, usually to around 100 000/µL, but thrombocytopenia is profound in some cases. Thrombocytope-nia alone does not usually cause serious bleeding and does not indicate severe malaria. Fibrinogen levels are usually elevated – a reduction indicates significant consumption (DIC). The fibrin degradation products are elevated. There is evidence of increased coagulation cascade activity through intrinsic pathway activation with antithrombin III depletion that is proportional to disease severity and there may be prolongation of the pro-thrombin and partial thromboplastin times. Polymorphonu-clear leucocyte elastase levels are elevated in severe infection, suggesting neutrophil activation.

The C-reactive protein, orosomucoid (α1-acid glycopro-tein), procalcitonin and fibrinogen levels are raised and immu-noglobulin levels rise while albumin falls. Cytokine levels are raised in acute malaria and there is an increase in urinary neop-terin. There may be mild hyponatraemia but the potassium is remarkably normal, unless there is severe acidosis, although it may fall during the recovery phase from severe malaria. The plasma bicarbonate is often reduced and the anion gap widens in proportion to the acidosis. The serum creatinine and blood urea may be raised, with often marked elevations in adults and an increased urea to creatinine ratio. Total and conjugated bili-rubin are often elevated in adults, the transaminase concentra-tions are often raised and there may also be slight elevation of the hepatic alkaline phosphatase concentration. In children the 5-nucleotidase is raised in proportion to disease severity. Cre-atinine phosphokinase, myoglobin and plasma urate levels are elevated in adults and children with severe malaria. The serum calcium may be low and hypophosphataemia may be profound in severe infections. Hypoglycaemia may occur and in the absence of quinine treatment this is accompanied by elevated ketones, raised plasma lactate and alanine and low insulin levels. Lactate levels in arterial or venous blood, or CSF, are elevated and blood bicarbonate is reduced in proportion to disease severity.

CEREBROSPINAL FLUID

The pressures in adults and children are similar, averaging approximately 160 mm CSF. But because the normal range in children is lower (<100 mm) most values in children are ele-vated. The CSF is usually normal in cerebral malaria, but mod-erately raised concentrations of protein are common (sometimes up to 200 mg/dL). There may be up to 10 cells/µL and on occa-sions up to 50 are seen (all lymphocytes). The CSF lactate con-centration is raised in cerebral malaria and the glucose may be slightly low relative to blood. If the patient is deeply jaundiced the CSF may appear yellow.

PROGNOSTIC FACTORS

The prognostic factors listed in Table 43.5 reflect vital organ dysfunction and the magnitude of the parasite burden. They are

In a proportion of cases there is bacterial septicaemia, but in the majority blood cultures are subsequently negative. In chil-dren poor capillary refill is a valuable prognostic sign. Shock usually responds temporarily to saline infusion and inotropes, but pulmonary oedema may be provoked if too much saline is given. The mortality is high. Orthostatic hypotension is common in acute uncomplicated malaria. It is associated with impaired reflex cardioacceleration and is worsened by the quinolone anti-malarial drugs. Rarely symmetrical peripheral gangrene can be associated with severe falciparum malaria. This does not appear to result from disseminated intravascular coagulation, but the role of red cell and platelet agglutination and vascular obstruc-tion have not been characterized.

HYPOGLYCAEMIA

Hypoglycaemia is either asymptomatic in severely ill patients, or presents as a further deterioration in the level of coma. It is a sign of poor prognosis. In severe malaria, the usual signs of sweating and increased sympathetic nervous system activity are commonly absent or indistinguishable from the signs of malaria. Hypoglycaemia occurs in approximately 8% of adults and up to 30% of children with cerebral malaria. It is often recurrent in quinine-treated patients. The clinical response to glucose is usually disappointing. In pregnant women with quinine-stimulated hyperinsulinaemic hypoglycaemia, the clinical fea-tures of hypoglycaemia are usually evident and the patient responds dramatically to glucose. Hypoglycaemia can often be prevented by infusion of 10% dextrose but frequent monitoring is still necessary.

ANAEMIA

The degree of anaemia and the rate at which it develops in malaria varies enormously. The haemoglobin concentration may fall by up to 2 g/dL each day. Anaemia is a particular problem in children, where profound anaemia may lead to sudden death. These complications are particularly likely with haemoglobin concentrations below 5 g/dL (15% haematocrit) and the risk rises steeply below 4 g/dL. Some patients appear to tolerate severe malarial anaemia relatively well. These patients usually have an underlying chronic anaemia and have adapted by increasing oxygen carriage (right-shifted oxygen dissociation curve). Thus it is both the absolute haemoglobin concentration and the magnitude of the fall that determine the clinical con-sequences. In the past a syndrome of malaria-associated anaemic congestive heart failure was often diagnosed and was managed by fluid restriction and often very cautious blood transfusion. It is now clear that the majority of these children with severe anaemia, rapid deep breathing and low blood pressure are aci-dotic and need quite the opposite treatment; intravenous rehy-dration and urgent blood transfusion.

PERSISTENT FEVER

Patients with severe malaria may have persistent fever after parasite clearance. Although a proportion of cases have an iden-tifiable chest or urinary tract infection, or in children blood cultures may grow Salmonella spp., the majority of cases have no clear explanation and the fever eventually resolves in a few days without further treatment.

566 SECTION 9 Protozoan Infections

because of quinine-stimulated hyperinsulinaemia. The concen-tration of lactate in venous or arterial blood or CSF is linearly proportional to the severity of disease. In terms of predictive prognostic value the admission venous bicarbonate concentra-tion has the best sensitivity and specificity and it is available widely. Persistent acidosis with low plasma bicarbonate and elevated plasma lactate four hours after admission indicates a poor prognosis. Although deep jaundice is often a bad sign, some adult patients develop a profound cholestatic jaundice without other evidence of vital organ dysfunction. Parasitaemia has traditionally been used as a measure of severity since the classic studies of Field and colleagues in Kuala Lumpur. They established that P. falciparum parasite counts over 100 000/µL were associated with an increased risk of dying and that the mortality of a count over 500 000/µL was 50%. The distribution of parasite counts in severe malaria is shifted to higher parasi-taemias in children living in areas of intense transmission, com-pared with non-immune adults. For example, parasite counts over 200 000/µL are not uncommon in ambulant semi-immune children who are mildly ill, whereas parasitaemias in this range are usually associated with severe disease in non-immune adults (Table 43.6). The sensitivity and specificity of parasitaemia alone as a prognostic indicator are limited, but can be improved by staging parasite development (more mature parasites – worse prognosis) and noting the number of polymorphonuclear neu-trophil leukocytes which contain pigment (>5% – poorer prog-nosis). For any parasitaemia the prognosis is worse if >20% of parasites contain visible pigment and better if >50% of parasites are at the tiny ring stage. In severe malaria, if >5% of neutro-phils contain visible pigment the prognosis is worse. Recent studies indicate that measurement of Plasmodium falciparum Histidine Rich protein2 (PfHRP2) in plasma or serum can be used to estimate the sequestered parasite biomass in severe malaria.

Antimalarial Drug TreatmentExtracts of the plant qinghao (Artemesia annua), known as qinghaosu, have been used in traditional medical practice in China for over two millennia. In AD340 Ge Hong described use of qinghao infusions for the treatment of fever in the famous Handbook of Emergency Treatments. Thereafter, qinghao is men-tioned frequently in the Chinese materia medica as a treatment for agues. Antimalarial drug discovery has often been linked to war. With the growing conflict in Vietnam in the 1960s Ho Chi Minh sought assistance from the Chinese leadership in combat-ing malaria threatening his troops. Chinese scientists examined both synthetic and traditional medicine treatments. The anti-malarial properties of qinghaosu were rediscovered in 1971 when extracts of the plant were shown to have activity against experimental rodent malaria. On the other side of the world another medicinal plant came to medical attention during the reign of the Count of Cinchon as Viceroy of Peru between 1628 and 1629 (Figure 43.19). Legend has it that the Viceroy’s wife, the Countess, was afflicted by ague in Lima. She was a well-known and popular figure and news of her illness spread inland. It eventually reached Lloxa where a Spaniard was in governor-ship. He knew of a local remedy obtained from the bark of a tree and sent it to the ailing Countess. The therapeutic result was excellent; she improved rapidly and was so impressed that she ordered the bark in quantity and dispensed it to the poor of Lima who commonly suffered from the dangerous tertian

not absolute and in fatal cases several factors usually co-exist. Some of the apparently poor prognostic factors can have a benign explanation. Hyperventilation (deep laboured breath-ing; respiratory distress) is usually a bad sign (indicating meta-bolic acidosis, pulmonary oedema, or pneumonia), but shallow tachypnoea can result from high fever alone (the tidal volume is lower). Upper gastrointestinal bleeding in cerebral malaria may also occur spontaneously. The prognostic implications of severe anaemia depend on the rate at which the haematocrit falls, the co-existing parasitaemia and metabolic abnormalities (particularly acidosis) and the stage of the infection. If anaemia develops gradually then even haemoglobin values <7 g/dL (packed cell volume <20%) can be surprisingly well tolerated as there is time for homeostatic adaptations such as the right shift in the oxygen dissociation curve, the increase in cardiac index and the fall in systemic vascular resistance. Hypotension is a poor prognostic sign only when associated with poor tissue perfusion, as evidence by cool peripheries and poor capillary refill. Patients, particularly children, with acute malaria often have very low blood pressures but they are warm and well per-fused. The biochemical measures are in general proportional to severity, but individual abnormalities can have other explana-tions. For example, hypoglycaemia carries a fivefold higher mortality in severe malaria, but in pregnant women treated with quinine hypoglycaemia may occur in uncomplicated infections

BIOCHEMISTRYHypoglycaemia <2.2 mmol/LHyperlactataemia >5 mmol/LAcidosis Arterial pH <7.3, venous plasma HCO3

<15 mmol/LSerum creatinine >265 µmol/la

Total bilirubin >50 µmol/LLiver enzymes sGOT (AST) >3 upper limit of normal

sGPT (ALT) >3 upper limit of normal5-Nucleotidase ≠

Muscle enzymes CPK ≠Myoglobin ≠

Urate >600 µmol/L

HAEMATOLOGYLeucocytosis >12 000/µL

Severe anaemia (PCV <15%)Coagulopathy Platelets <20 000/µL

Prothrombin time prolonged >3 sProlonged partial thromboplastin timeFibrinogen: <200 mg/dL

PARASITOLOGYHyperparasitaemia >100 000/µL – increased mortalityb

>500 000/µL – high mortalityb

>20% of parasites are pigment-containing trophozoites and schizonts

>5% of neutrophils contain visible malaria pigment

PCV, packed cell volume; sGOT (AST), serum glutamic oxaloacetic transferase (aspartate aminotransferase); sGPT (ALT), serum glutamic pyruvic transaminase (alanine aminotransferase); CPK, creatine phosphokinase.

aThis is the criterion for adults. Less elevated values are found in children with severe malaria.

bThese refer to thresholds in non-immune adults. The thresholds are much higher in children in endemic areas.

TABLE 43.5

Laboratory Indicators of a Poor Prognosis in Severe Malaria

43  Malaria 567

fever, his personal physician Jean-Jacques Chifflet began a bitter polemic on the merits of the bark, which was to last for 200 years. Much of the dispute stemmed from the fact that many considered all fevers had the same cause and clearly not all responded to Jesuit’s bark. It was probably Torti in 1712 who first stated that the bark was ‘specific solely for the ague’.

Another source of debate and one that is still active today, was dosage. Sir Robert Talbor [Talbot] was one of the few physi-cians who was not afraid to give the bark in large and repeated doses and when he cured the Dauphin (the son of Louis XIV) with his remede anglais his fame spread far and wide. He subsequently treated Charles II of England successfully with the same medicament. Others were less enthusiastic. Many

fevers. The pulverized bark became known as ‘los polvos de la Condeca’ or the Countess’s powder and Linnaeus subsequently named the tree from which the bark was obtained ‘Cinchona’ in honour of the Countess. Sadly, the detective work of one AW Haggis, reported in 1941, has shown that ‘the fabulous story of the Countess of Cinchon’ is almost certainly a romantic fable. Nevertheless, it is likely that the bark was introduced to Europe by the Fathers of the Society of Jesus around the time of the story, or even earlier (c.1630) and was widely promoted in Europe by the Jesuit Cardinal Juan de Hugo. For these reasons it became known as Jesuit’s bark. Not everyone was convinced by the new remedy and when in 1653 Archduke Leopold of Austria relapsed 1 month after being cured of double quartan

Prognostic valuea Frequencya

Children Adults Children Adults

CLINICAL MANIFESTATIONSB

+ (?) Prostration +++ ++++++ ++ Impaired consciousness +++ +++++ +++ Respiratory distress (acidotic breathing) +++ +++ ++ Multiple convulsions +++ +

+++ +++ Circulatory collapse + ++++ +++ Pulmonary oedema (radiological) ± ++++ ++ Abnormal bleeding ± +++ + Jaundice + ++++ + Haemoglobinuria ± +

LABORATORY FINDINGS+ + Severe anaemia +++ +

+++ +++ Hypoglycaemia +++ +++++ +++ Acidosis +++ +++++ +++ Hyperlactataemia +++ ++± ++ Hyperparasitaemia ++ +++ ++ Renal impairment + +++

aOn a scale from + to +++ ; ± indicates borderline prognostic value or infrequent occurrence.bAnuria and hypothermia (core temperature <36.5°C) are also poor prognostic signs.

TABLE 43.6 Severe Manifestations of P. falciparum Malaria in Adults and Children

Figure 43.19 (A) The ‘Arbor febrifuga Peruviana’. ‘In the district of the city of Loja, diocese of Quito, grows a certain kind of large tree, which has a bark like cinnamon, a little more coarse and very bitter: which ground to powder is given to those who have fever and with only this remedy, it leaves them’ (A, Bernabe Cobo S. J. Historia del Nuevo Mundo; 1582–1657.) (B) Qinghao (Artemisia annua) plantation in China.

A B

568 SECTION 9 Protozoan Infections

4-aminoquinoline, chloroquine, in 1934. Initially, chloroquine was rejected as being too toxic for human use and the research team at Bayer were asked to produce a safer compound. They then produced 3-methylchloroquine (Sontoquine) but, despite clinical studies, these compounds were generally unavailable at the outbreak of the Second World War.

Armies fighting in tropical theatres of war usually lose more men to malaria than bullets. At the outset of the Second World War, the Allies knew their position was precarious in the tropics as most of the world’s Cinchona was grown in Java and this was vulnerable to Japanese invasion. They embarked upon a tre-mendous combined research effort into the development and evaluation of new antimalarials. This led to the rediscovery of chloroquine and the development of primaquine. An entirely separate line of research in the UK led to the discovery in 1945 of the antimalarial biguanides, proguanil and subsequently chlorproguanil. These compounds were later shown to inhibit the plasmodial enzyme dihydrofolate reductase (DHFR). Researchers at the Wellcome Research Laboratories synthesizing purine analogues developed the antimitotic compound 6-mercaptopurine (and later azathioprine) and in 1952 discov-ered the antiprotozoal DHFR inhibitor pyrimethamine. This same line of Nobel Prize winning research later developed tri-methoprim, which has considerably greater affinity for bacterial DHFR (but also inhibits the plasmodial enzyme) and also allo-purinol, acyclovir and zidovudine (AZT).

By the early 1950s, the 4-aminoquinolines, chloroquine and to a much lesser extent amodiaquine, had become the treatment of choice for all malaria throughout the world. Pyrimethamine was also used in treatment and chloroquine, pyrimethamine and proguanil were used for prophylaxis. Primaquine was given to prevent relapses of P. vivax and P. ovale. The Cinchona alka-loids were little used outside Francophone Africa and, with the discontinuation of quinine, blackwater fever became a rarity. This was the heyday of the malaria eradication era and with the tremendous successes in Europe and North America and many urban areas of the tropics, interest in the development of new antimalarial drugs waned rapidly. But eradication in the tropics failed and in the 1960s antimalarial drug resistance emerged as a major threat.

Until the early 2000s most countries relied on chloroquine to treat malaria and when this failed they turned to sulfadoxine-pyrimethamine (SP). But resistance to chloroquine emerged at the end of the 1950s simultaneously in Colombia and the Thai–Cambodian border and over the next four decades spread across the entire tropical world. The expanding tide of antimalarial drug resistance, together with the looming conflict in Vietnam and the manifest failure of the eradication programme prompted a massive US army-led research effort to screen and test new antimalarial compounds. Most of the compounds developed were structurally related to the known quinoline antimalarials (mefloquine, halofantrine, tafenoquine). In the 1980s, the hydroxynaphthaquinone compound atovaquone (a modifica-tion of a compound discovered over 50 years ago) was com-bined with proguanil in a single fixed-dose formulation which is a safe and highly effective antimalarial, but is very expensive to manufacture.

It is the Chinese who have given us the most important antimalarial drugs in recent years. Four are related to quinoline antimalarials; lumefantrine (formerly known as benflumetol), pyronaridine, piperaquine and naphthoquine and all are active against multi-drug-resistant malaria. By far the most important

Protestants believed the bark to be a poison disseminated by the Jesuits. The dose–response question was clarified in 1768 by Lind, who demonstrated clearly that in order to get best results the bark should be given in full doses as soon as the disease was diagnosed (advice that has stood the test of time).

In 1820, the French chemists Pierre Pelletier and Joseph Caventou isolated the alkaloid quinine from cinchona bark. Purification of the various cinchona alkaloids allowed stan-dardization of dosage. Adequate doses could now be given in relatively small amounts of pure drug, but by the middle of the nineteenth century enormous doses (up to 100–150 grains over 2 days) were being prescribed. Toxicity was common and the popularity of the medicine fell. Gradually, however, the diagno-sis of agues and the prescription of Cinchona alkaloids became more rational and logical. The new colonial powers recognized the importance of Cinchona and improved methods of horti-culture resulted in better yields of the alkaloids from the culti-vated trees. The Dutch took the lead and vast plantations of high-yielding Cinchona ledgeriana were started in the East Indies (principally in Java).

Laveran, having identified haematozoa as the cause of palu-dism, later concluded that quinine cured the disease by killing the newly discovered parasites. This theory encountered con-siderable resistance in the years immediately following its pub-lication. In 1880 Bacelli described the intravenous method of administering quinine (although there is evidence that this route had been used for 50 years before that). Laveran consid-ered intravenous injection to be dangerous, giving rise to both local and general complications and was only justified in ‘the most grave and pernicious disease’. He also confirmed the earlier observations of Thomas Willis (1659) that cinchona cured the acute attacks of ague, but did not prevent relapses and also appeared to have no effect on crescents (gametocytes of P. falciparum). The eminent Italian malariologists subse-quently showed that quinine prevented asexual blood-stage development but could not stop sporulation of formed seg-menters (meronts).

In England in 1856, William Henry Perkin accidentally dis-covered analine purple (mauve) while attempting to synthesize quinine from coal tar products. Thus began the synthetic dye industry. Later in Germany, the antimicrobial properties of those newly discovered aniline dyes were investigated. In 1890 Ehrlich showed that methylene blue had antimalarial activity against P. cathemerium in canaries, but the dye proved disap-pointing in clinical practice (although it is now undergoing a resurgence of interest) and structural modifications did not lead to compounds with improved activity. During the Great War (1914–1918), whole armies were immobilized in the Balkans because of malaria and there were heavy losses in Mesopotamia, East Africa and the Jordan Valley. The British and French armies used quinine extensively and despite frequent objections to the bitter medicament, many lives were saved. The military and strategic importance of antimalarial drugs stimulated much research immediately after the war. In the early 1920s the resur-gent German chemical industry again focused its attention on new antimicrobial compounds. The first synthetic antimalarial was discovered in 1924 originating from attempts to combine chemically the properties of quinine and methylene blue. This was an 8-aminoquinoline compound, plasmoquine, also known as pamaquine or plasmochin, a precursor of primaquine. Plasmoquine was followed by the acridine compound mepa-crine (atebrine, quinacrine) in 1932 and the structurally related

43  Malaria 569

parasites and events there act as harbingers of the development of resistance elsewhere (Figure 43.20). Chloroquine resistance spread throughout the region in the 1960s and 1970s and SP fell rapidly to resistance in the early 1980s. In 1984 mefloquine replaced quinine as the treatment of choice for falciparum malaria in Thailand. This was the first country in which meflo-quine was used widely. It was introduced in combination with sulfadoxine and pyrimethamine in order to delay the onset of resistance. However, since 1988 mefloquine resistance devel-oped rapidly in Thailand and adjacent Cambodia and western Burma and later in Vietnam, while sensitivity to quinine declined very gradually. By 1994 high-level mefloquine resis-tance had developed in some areas with early treatment failures in 10% of cases. On the western border of Thailand, the combination of artesunate, given for 3 days and high-dose mefloquine (25 mg/kg) was introduced. Despite the fact that P. falciparum there was already mefloquine-resistant, this proved remarkably effective. In the subsequent 15 years cure rates remained over 90%. Following deployment of the ACT there was an improvement of mefloquine sensitivity and there was a marked decline in the incidence of falciparum malaria. Unfor-tunately, resistance to the artemisinins has emerged in western Cambodia. By 2007 there was increasing evidence of reduced susceptibility and by 2009 there was unequivocal proof29 (Figure 43.21). Resistance has also either spread or emerged independently on the Thailand–Myanmar border and in south-ern Vietnam. Resistance is manifest by considerable slowing in the rate of parasite clearance following artemisinin treatment. This has now begun to compromise the efficacy of ACTs, par-ticularly artesunate-mefloquine. Cure rates on the western border of Thailand have fallen from over 90% to less than 70% in the past 2 years. Up to date information on all aspects of antimalarial drug resistance is freely available on the internet (http://www.wwarn.org).

development in malaria treatment in recent years has been the Chinese rediscovery and development of the drugs related to artemisinin (qinghaosu). They are structurally unrelated to existing antimalarials, rapidly effective, well tolerated and safe. After an inordinate delay in gaining global recognition, artemis-inin-based combination treatments (ACTs) are now recom-mended by the World Health Organization as first-line treatment for all uncomplicated falciparum malaria and paren-teral artesunate is the treatment of choice for severe malaria. There have been more clinical trials on artemisinin and its derivatives than any other antimalarial drugs.

Between the 1960s and the 1990s there was very little research on new antimalarial drugs by the international phar-maceutical industry. In recent years, increased international funding and the formation of public–private partnerships has lead to a resurgence of research and development. There is now the ‘healthiest’ pipeline for new antimalarials in living memory and over 15 new antimalarials are in various stages of development.

ANTIMALARIAL DRUG RESISTANCE

In the last two decades of the twentieth century, the global death toll from malaria rose while the mortality from other infectious diseases (with the notable exception of HIV-AIDS) generally fell. This was attributed directly to drug resistance. Plasmodium falciparum has now developed resistance to all classes of anti-malarial drugs including the artemisinin derivatives. The other human malarias are generally more sensitive to antimalarial drugs than P. falciparum, although resistance of P. vivax to antifols is widespread. Significant chloroquine resistance has now also developed in Plasmodium vivax in many locations. Quinine resistance in P. falciparum was first reported from Brazil in 1910, but has never been high grade and has not com-promised use of the drug. Within years of the introduction of the antifols proguanil and pyrimethamine, resistance was noted in both P. falciparum and P. vivax which certainly did compro-mise use of these drugs, but antimalarial resistance was not treated seriously until chloroquine resistance in P. falciparum developed almost simultaneously in South-east Asia and South America at the end of the 1950s. The selection of resistance may have resulted from the misguided use of chloroquine (and pyri-methamine) impregnated salt in an attempt to control malaria by mass prophylaxis. During the 1970s, chloroquine resistance in P. falciparum spread from South-east Asia and South America and fuelled the resurgence of falciparum malaria in the tropics. By the early 1980s chloroquine was no longer effective in many countries and the first ominous reports of resistance from the east coast of Africa appeared. Since then chloroquine resistance spread remorselessly across Africa and today few countries in the tropics (such as those north of the Panama Canal) are unaf-fected. Pyrimethamine resistance has also worsened rapidly and the synergistic combination with sulphonamides (SP; sulphadoxine-pyrimethamine) is no longer effective in much of East Asia, Southern and Central Africa and South America. The importance of transcontinental spread of resistance in P. falci-parum has been highlighted by recent molecular epidemiologi-cal studies which confirm that both the chloroquine resistance and the SP resistance that have wreaked such havoc in Africa, originated in South-east Asia.6

South-east Asia and in particular Cambodia and Thailand have traditionally had the world’s most drug-resistant malaria

Figure 43.20 Antimalarial resistance at its most severe; the decline in antimalarial drug efficacy against P. falciparum malaria on the western border of Thailand. Chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) were no longer effective by the beginning of the 1980s. Quinine (Q) efficacy declined slowly. Mefloquine (M) was introduced in Novem-ber 1984 (arrow), at a dose of 15 mg/kg combined with SP–although SP was already useless by then. Resistance to mefloquine developed rapidly despite tight control over its use. A 7-day quinine-tetracycline regimen (QT) remained effective, but the introduction of artesunate-mefloquine (artesunate 12 mg/kg over 3 days, combined with 25 mg/kg mefloquine split dose) in 1994 led to a remarkable reversal of the resistance trend. This regimen remained over 90% efficacious until 2010, after which efficacy declined sharply coincident with the increasing prevalence of artemisinin-resistant parasites.

1975 1980 1985 1990 1995 2000 2005 2010

Cur

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)

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20

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CQ

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QT A3M25

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M15 M25

570 SECTION 9 Protozoan Infections

to a negative figure from −10 to −10 000, thus reducing parasite numbers by between 10- and 10 000-fold per cycle. The Emax is the maximum effect, which is the effect represented at the top of the sigmoid dose–response or concentration–effect relation-ship. Drugs differ in their Emax; for example the artemisinins often produce a 10 000-fold reduction per asexual cycle, whereas antimalarial antibiotics such as tetracycline or clindamycin may achieve only at most a 10-fold parasite reduction per cycle (Figure 43.22). The lowest blood or plasma concentration of antimalarial drug which results in Emax can be considered a minimum parasiticidal concentration or MPC. Parasite reduc-tion appears to be a first-order process throughout. This means that provided that the MPC is exceeded then a fixed fraction of the population is removed each successive asexual cycle. Patients with acute malaria may have up to 1012 parasites in the circula-tion. Even with killing fractions per cycle of 99.99% it will take at least three life cycles (6 days) to eradicate all the parasites. Thus antimalarial treatment must usually provide therapeutic drug concentrations for 7 days (covering four cycles) to effect a cure. For rapidly eliminated drugs this means the course of treatment must be 7 days (Figure 43.23). Treatment responses are always better in patients with some immunity. In endemic areas this means that the worst treatment results are seen in young children.

In the treatment of severe malaria the antimalarial drug activity on the different stages of parasite development is also important as the object of treatment is to stop parasite matura-tion, particularly from the less pathogenic circulating ring forms to the more pathogenic cytoadherent stages. The drugs used for the treatment of severe malaria all act predominantly in the middle third of the life cycle when there is the greatest increase in parasite synthetic and metabolic activity. The anti-fols act later on the forming schizont, but none of the drugs will prevent rupture and reinvasion once the meront (schizont) has formed (the widely used term schizontocidal is therefore

ANTIMALARIAL TREATMENT

In general, the antimalarial drugs are more toxic than antibac-terials, i.e. the therapeutic ratio is narrower, but serious adverse effects are rare. The available antimalarials fall into three broad groups: the aryl aminoalcohol (quinoline-related or quinoline-like) compounds (quinine, quinidine, chloroquine, amodia-quine, mefloquine, halofantrine, lumefantrine, piperaquine, pyronaridine, primaquine, tafenoquine); the antifols (pyri-methamine, proguanil, chlorproguanil, trimethoprim); and the artemisinin compounds (artemisinin, dihydroartemisinin, arte-mether, artemotil, artesunate). Of these, the artemisinin drugs have the broadest time window of action on the asexual malar-ial parasites, from ring forms to early schizonts, and they produce the most rapid therapeutic responses. It is the unusual ring stage activity which explains their rapidity of action and their life-saving benefit in severe malaria and it is this property which is lost in resistant parasites. Several antibacterial drugs also have antiplasmodial activity, although in general their action is slow and they are used in combination with the anti-malarial drugs. Those used are the sulphonamides and sul-phones, tetracyclines, clindamycin, macrolides and inadvertently, chloramphenicol. Fosmidomycin is an active antimalarial anti-biotic under investigation. Significant resistance has been reported to the sulphonamides but not the other classes of antibiotics (although macrolide resistance is readily induced in the laboratory). Drugs which are active against sensitive P. fal-ciparum are also active against the other malaria species.

Antimalarial PharmacodynamicsThe principal effect of antimalarial drugs in the treatment of uncomplicated malaria is to inhibit parasite multiplication (by stopping parasite development). The untreated infection can multiply at a maximum rate given by the average number of viable merozoites per mature schizont (100% efficiency). In non-immunes multiplication is often relatively efficient with multiplication rates of 6–20/cycle (30–90% efficiency). Antima-larials exerting their maximum effects (Emax) will convert this

Figure 43.21 Artemisinin-resistant P. falciparum malaria; parasite clearance profiles in Western Cambodia (Pailin) where resistance was established and Western Thailand (Wang Pha) following artesunate monotherapy (AS7) and mefloquine–artesunate (MAS3).29

10-3

10-2

10-1

100

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103

Time (h)

12 24 36 48 60 72 84 96 108 120

Para

sita

emia

(% fr

om a

dmis

sion

, med

ian,

IQR

) Pailin AS7 Pailin MAS3 Wang Pha AS7 Wang Pha MAS3

Figure 43.22 Pharmacodynamics. The effects of different rates of parasite killing on the elimination of the malaria infection. The individual parasite biomass is given on the vertical axis. 1012 parasites corresponds to about 2% parasitaemia in an adult who is not anaemic. The artemis-inin derivatives achieve the highest parasite reduction rations (PRR: 104 per asexual cycle) and eradicate the infection in 6–8 days. Most of the other antimalarials achieve PRR values of 102–103 per cycle. The antima-larial antibiotics alone (e.g. doxycycline) have PRR values of approxi-mately 10 and take 3 weeks to cure malaria if used alone (which they clearly should not be!).

Tota

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43  Malaria 571

mutation at position 164 (isoleucine to leucine). In Plasmodium vivax a similar sequence of events occurs with sequential acqui-sition of mutations in PvDHFR conferring increasing antifol resistance. The I164 L mutation renders the available antifols completely ineffective against Plasmodium falciparum malaria. This mutation is prevalent in parts of SE Asia and South America and has been reported in East Africa. Interestingly mutations conferring moderate pyrimethamine resistance do not necessarily confer cycloguanil resistance and vice versa. For example, mutations at position 16 (alanine to valine) plus serine to threonine at 108 confer high-level resistance to cyclo-guanil but not pyrimethamine.5 In general the biguanides (cyc-loguanil, chlorcycloguanil) are more active than pyrimethamine against the resistant mutants (and they are more effective clini-cally too), but they are ineffective against parasites with the Pfdhfr I164L mutation.

The marked synergy with sulphonamides and sulphones is very important for the antimalarial activity of sulpha-pyrimethamine or sulphone-biguanide combinations. Sul-phonamide and sulphone resistance also develops by progressive acquisition of mutations in the gene encoding the target enzyme DHPS (which is a bifunctional protein with the enzyme PPPK). Specifically in Plasmodium falciparum altered amino acid resi-dues associated with reduced antifol susceptibility have been found at positions 436, 437, 540, 581 and 613 in the DHPS domain. Parasites with DHPS mutations nearly always have DHFR mutations as well. The addition of the 540 to the 437 mutation is associated with particularly high failure rates. Plasmodium falciparum parasites with ‘quintuple mutations’ (Pfdhfr S108N, N51I and C59R and Pfdhps A437G, K540E) are now widespread in tropical countries and are associated with high SP treatment failure rates and poor responses to the arte-sunate SP combination. The Pfdhps 581 and 631 mutations are also increasingly prevalent. They do not occur in isolation, but always on top of an initial mutation (usually alanine to glycine at 437) and confer additional resistance.

Quinolines and Related DrugsThe mode of action of the quinoline antimalarials has been a source of controversy for years. These drugs are weak bases and they concentrate in the acid food vacuole of the parasite, but this in itself does not explain their antimalarial activity. Chloroquine intercalates DNA, but only at concentrations (1–2 mmol/L) much higher than required to kill parasites (10–20 nmol/L). Chloroquine binds to ferriprotoporphyrin IX, a product of haemoglobin degradation and thereby chemically inhibits haem dimerization. This is an essential defence mecha-nism for the parasite to detoxify haem and inhibition of this process provides a plausible explanation for the selective anti-malarial action of these drugs. Chloroquine also competitively inhibits glutathione-mediated haem degradation, another para-site detoxification pathway. Chloroquine resistance is associated with reduced concentrations of drug in the acid food or diges-tive vacuole. Both reduced influx and increased efflux have been implicated. The resistant parasites lose chloroquine from the digestive vacuole 40–50 times faster than drug-sensitive para-sites. This efflux mechanism is similar to that found in multi-drug-resistant (MDR) mammalian tumour cells. The first efflux mechanism to be characterized was the ATP-requiring trans-membrane pump, P. glycoprotein. These unmutated Pfmdr1 genes are found in increased copy numbers in most quinine-,

incorrect). Young rings are also relatively drug-resistant (par-ticularly to quinine and pyrimethamine). The artemisinin com-pounds have the broadest time window of antimalarial action and the most rapid in vivo activity. These compounds and to a lesser extent chloroquine, prevent maturation of ring stages inducing accelerated clearance and reducing subsequent cytoad-herence (whereas quinine does not). The life-saving benefit of artesunate over quinine in severe malaria results largely from the additional killing of circulating ring stage parasites which are thereby prevented from maturing and sequestering. Quinine does not do this.

MODE OF ACTION AND MECHANISMS OF RESISTANCE

Resistance means that there is a shift to the right in the dose–response (concentration–effect) relationship. Higher concen-trations of drug are required to achieve parasite killing. The shape of the relationship may also change and the maximum effect lowered.

Antifols and SulphasPyrimethamine and the antimalarial biguanides interfere with folic acid synthesis, in the parasite by inhibiting the bifunctional enzyme dihydrofolate reductase – thymidylate synthase (DHFR). Sulphonamides act at the previous step in the syn-thetic pathway by inhibiting dihydropteroate synthase (DHPS). There is marked synergy in antimalarial activity between the two classes of compounds. Resistance to proguanil and pyri-methamine in P. falciparum and P. vivax were reported within a few years of their introduction. DHFR resistance is associated with point mutations in the DHFR gene which lead to reduced affinity (100–1000 times less) of the enzyme complex for the drug. The first mutation is usually at position 108 of Pf DHFR (serine to asparagine) and the corresponding position 117 of PvDHFR. This has little clinical relevance for treatment initially, but then in P. falciparum, mutations arise at positions N51I and C59R conferring increasing levels of in vivo resistance to pyri-methamine. Infections with triple mutants are relatively resis-tant but some therapeutic response is usually seen. The stage is then set for the acquisition of a fourth and devastating

Figure 43.23 Pharmacokinetics. A comparison of the elimination pro-files of the antimalarial drugs (normalized to the initial peak concentra-tion). A, artemisinin and derivatives; Qn, quinine/quinidine; Pyr, pyrimethamine; Lum, lumefantrine; Cq, chloroquine; Mef, mefloquine; Pip, piperaquine.

Weeks

AQn

Pyr Lum

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Plas

ma

conc

entra

tion

(%)

572 SECTION 9 Protozoan Infections

THE EMERGENCE AND SPREAD OF ANTIMALARIAL DRUG RESISTANCE

Antimalarial drug resistance is a major threat to health in the tropics. The development of resistance can be divided into the relatively rare event giving rise to resistance de novo and the subsequent spread of resistance among the parasite population. Malaria parasites do not acquire resistance genes by lateral transfer from other parasites. Resistance arises from spontane-ous chromosomal point mutations or gene duplications which are independent of drug selection pressure. Once formed, these more resistant mutants have a survival advantage in the pres-ence of antimalarial drugs. Several factors encourage the devel-opment of resistance. These are the intrinsic frequency with which the genetic changes occur, the degree of resistance con-ferred by the genetic change (pharmacodynamics), the propor-tion of all transmissible infections which are exposed to the drug, the drug concentration profile (pharmacokinetics), the pattern of drug use and the immunity profile of the community. Resistant parasites will be selected when parasites are exposed to subtherapeutic drug concentrations (i.e. concentrations which would eradicate most sensitive infections but not infec-tions with the resistant mutants). Thus, non-immune patients infected with large numbers of parasites who receive inadequate treatment (either because of poor drug quality, adherence, vom-iting of an oral treatment, etc.) are a potent source of de novo resistance. This emphasizes the importance of correct prescrib-ing and good adherence to prescribed drug regimens, particu-larly in patients with heavy parasite burdens in slowing the emergence of resistance.

The emergence of resistance is slower in high transmission areas, because background immunity eliminates the majority of infections and so clears resistant mutants and stops them being transmitted. The spread of resistant mutant parasite is facili-tated by the use of drugs with long elimination phases which provide a ‘selective filter’, allowing infection by the resistant parasites while the residual antimalarial activity prevents infec-tion by sensitive parasites. Slowly eliminated drugs such as mefloquine (T1/2 β 2–3 weeks) or chloroquine (T1/2 β 2 months) persist in blood and provide such a selective filter for months after drug administration (Figure 43.23). The selection

mefloquine- and lumefantrine-resistant P. falciparum parasites, whereas point mutations in codons 86 (N86Y) and also 184 (Y184F) and 1246 (D1246Y) have been related to chloroquine and amodiaquine resistance. Amplification of Pfmdr is the main contributor to mefloquine and lumefantrine resistance. Trans-fection studies confirm a role for Pfmdr in mediating resistance to chloroquine and mefloquine. Thus lumefantrine and amo-diaquine, the two most widely used partner drugs in Africa provide opposite selection pressures on Pfmdr so deployment of both together would be expected to slow the emergence of resistance at this locus. But the critical discovery has been the association of point mutations in CRT (a food vacuolar mem-brane protein thought to have a transporter function), with chloroquine resistance. The central role of a PfCRT mutation resulting in a change in coding from lysine to threonine at posi-tion 76 gene in mediating chloroquine resistance has been shown unequivocally in the laboratory by transfection studies and in epidemiological studies where therapeutic responses are predicted by this single polymorphism. In several regions where chloroquine resistance was prevalent, a reversion to wild-type PfCRT in parasite populations has been associated with a return of chloroquine susceptibility.30 PfCRT also plays an important role in amodiaquine and quinine resistance. From an epidemio-logical standpoint multiple unlinked mutations probably con-tribute to chloroquine resistance, modifying the central role of CRT. It is likely that other contributors to quinoline resistance remain to be discovered.

The chloroquine efflux mechanism in resistant parasites can be inhibited by a number of structurally unrelated drugs: calcium channel blockers, tricyclic antidepressants, phenothi-azines, cyproheptadine, antihistamines, etc. whereas meflo-quine resistance is reversed by penfluridol, which does not reduce chloroquine efflux. This gave hope that chloroquine resistance might be reversed in clinical practice. Initial evalua-tions were uniformly disappointing, but studies in Nigerian children given chloroquine together with very high doses of chlorpheniramine, did indicate significantly improved efficacy against chloroquine-resistant falciparum malaria. In general antimalarial drug resistance to mefloquine, quinine, lumefan-trine and halofantrine is linked. Within a particular geographic area there is a reciprocal relationship; increasing mefloquine resistance is associated with increasing susceptibility to chloroquine.

AtovaquoneAtovaquone interferes with parasite mitochondrial electron transport and it also depolarizes the parasite mitochondria thereby blocking cellular respiration. High levels of resistance result from single point mutations in the gene encoding cyto-chrome b. This gene is encoded on a small extrachromosomal plastid-like DNA-containing organelle (the apicoplast), which is phylogenetically of algal origin. Resistance mutations arise frequently in vitro and in vivo (Figure 43.24).

Artemisinin and DerivativesThe mechanism of action of the artemisinin drugs remains uncertain. Initially it was thought to involve cation (mainly the ferrous ion) mediated generation of carbon-centred free radi-cals which alkylate critical proteins. Parasiticidal activity is cer-tainly dependent on the integrity of the peroxide bridge and can be blocked by the iron chelator desferrioxamine.

Figure 43.24 The de novo emergence of high-level resistance. Fol-lowing atovaquone (only) treatment highly resistant recrudescent infec-tions emerged in one-third of patients. This represented untrammelled growth of a single starting parasite; a per parasite mutation frequency of 1 in 1012.

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43  Malaria 573

lemon) and it is an effective treatment for night cramps, as well as for malaria. Contrary to widespread belief quinine is not antipyretic. Quinine is usually formulated as the dihydrochlo-ride salt for parenteral administration and as the sulphate, bisulphate, dihydrochloride, ethylcarbonate, hydrochloride or hydrobromide salts for oral administration. Unlike the other antimalarials and somewhat confusingly, quinine doses are usually prescribed as weights of salt rather than base (the dif-ferent salts have different base contents). Quinine acts princi-pally on the mature trophozoite stage of parasite development. It does not prevent sequestration or further development of formed meronts and does not kill the pre-erythrocytic or sexual stages of P. falciparum.

PharmacokineticsQuinine is well absorbed after oral or intramuscular adminis-tration both in adults and children (Table 43.7). Peak levels are usually reached within 4 hours (more rapidly if the intramus-cular injections are diluted) (Figure 43.23). In acute malaria the total apparent volume of distribution (Vd) is contracted and systemic clearance reduced in proportion to disease severity. As a result blood concentrations are higher in uncomplicated malaria than in healthy subjects and highest in severe malaria. The elimination half-life is approximately 18–20 hours in cere-bral malaria, 16 hours in uncomplicated malaria and 11 hours in health. In children and pregnant women the apparent volume of distribution is relatively smaller and elimination is more rapid. Malnutrition reduces both Vd and clearance similar to malaria. Quinine is a base and is bound principally to the acute phase plasma protein α1-acid glycoprotein. Plasma protein binding is increased in malaria from approximately 75–80% in healthy subjects to over 90% in severe malaria. Red cell concen-trations vary between one-third and one-half of corresponding plasma concentrations and concentrations in breast milk and cord blood are approximately one-third of those in plasma. The therapeutic range has not been well defined but total plasma concentrations of between 8 and 15 mg/L are certainly safe and effective. Toxicity is increasingly likely with plasma concentra-tions over 20 mg/L (free quinine >2 mg/L). Approximately 80% of the administered drug is eliminated by hepatic biotransfor-mation, principally via CYP 3A4 and also CYP 3A5 and the remaining 20% is excreted unchanged by the kidney. Although systemic clearance is reduced in severe malaria, this 80 : 20 pro-portion is preserved. The principal metabolite 3-hydroxyquinine is biologically active, contributing approximately 10% to anti-malarial activity, but more in renal failure where it accumulates. The other more polar metabolites are either much less active, or inactive as antimalarials.

ToxicityMinor adverse effects are common with quinine but serious toxicity is remarkably rare in the treatment of malaria. Allergic reactions (thrombocytopenia, haemolysis, rash, haemolytic-uraemic syndrome) are all rare in malaria treatment. Quinine is extremely bitter and therefore unpleasant to take and regu-larly produces a symptom complex known as ‘cinchonism’. This comprises tinnitus, reversible high-tone hearing impairment, nausea, dysphoria and often vomiting. As a consequence com-pliance with the 7-day regimens required for cure is poor. Quinine predictably prolongs depolarization in skeletal and cardiac muscle and this is the main contributor to the prolonga-tion of the QTc interval on the electrocardiograph by

pressure can be enormous. In Africa approximately 250 000 kg or 170 × 106 adult treatment doses of chloroquine were con-sumed annually. Thus in many parts of the continent the major-ity of the population had chloroquine in the blood at any time.

The emergence of resistance can be prevented by the use of combinations of drugs with different mechanisms of action and therefore different drug targets. The same rationale underlies the current treatment of tuberculosis, leprosy, HIV infections and many cancers. If two drugs are used, which do not share a common mode of action and therefore the parasite develops different mechanisms of resistance to them, then the per-parasite probability of developing resistance to both drugs is the product of their individual per-parasite probabilities. For example, if the per-parasite probabilities of developing resis-tance to drug A and drug B are both 1 in 10,12 then a simultane-ously resistant mutant will arise spontaneously every 1 in 1024 parasites (Figure 43.25). The lower the de novo per-parasite probability of developing resistance, the greater the delay in the emergence of resistance. However, this powerful approach has several limitations. If not everyone receives the combination and some patients only receive one of the components, then resistance can arise (emphasizing the importance of achieving high coverage when these drugs are deployed). Mutual protec-tion works only if the both drugs are always present together, but in current ACTs there is a considerable pharmacokinetic mismatch such that the slowly eliminated partner drug is present for days or weeks unprotected by the artemisinin com-ponent. This will enhance the spread of resistance. Combina-tions are also more expensive. But the increased cost is outweighed by the longer-term benefits. Another effective approach to delaying the spread of resistance is to deploy dif-ferent drugs with different resistance mechanisms at the same time. With this ‘multiple first-line treatment’ approach the fitness disadvantage incurred by resistance mechanisms is exploited. At present there are a number of important, but unanswered, practical questions concerning resistance. We do not know the relative importance of all the factors which con-tribute and therefore the optimum strategy to prevent resistance.

QUININE

Quinine is a bitter powder obtained from the bark of the Cin-chona tree. It is widely used as a flavouring (tonic water, bitter

Figure 43.25 The logarithmic distribution of malaria parasites. There are probably between 1016 and 1018 parasites in the world today. The day after tomorrow, there will be another 1016–1018!

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574 SECTION 9 Protozoan Infections

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43  Malaria 575

therapeutic levels are reached as quickly as possible. If adequate treatment has been given before referral to hospital (i.e. >15 mg/kg in the preceding 24 hours) the loading dose is unnecessary. But if there is any doubt at all, or lower doses have been given, then the full loading dose should be given. In severe malaria quinine doses should be reduced by one-third to one-half after 48 hours if there is no clinical improvement, or if there is acute renal failure. This prevents blood concentrations accumulating to toxic levels. Intramuscular quinine is painful and sclerosant if given undiluted (300 mg/mL). It should be diluted in sterile water 1 : 3 to 1 : 5 and given to the anterior thigh, never the buttock (to avoid the risk of sciatic nerve damage), using strict aseptic technique. Oral quinine is given in a dose of 10 mg salt/kg three times daily for 7 days (shorter courses are less effective) combined with either doxycycline or clindamycin.

CHLOROQUINE

Chloroquine is a 4-aminoquinoline. It is formulated as sul-phate, phosphate and hydrochloride salts and is prescribed in weights of base content. Various liquid formulations are avail-able for paediatric use. Chloroquine can be given by intrave-nous infusion, intramuscular or subcutaneous injection, orally, or by suppository. Chloroquine acts mainly on the large ring-form and mature trophozoite stages of the parasite. It produces more rapid parasite clearance than quinine but is slower than artemisinin derivatives. Chloroquine is also used in the treat-ment of hepatic amoebiasis and for some collagen-vascular and granulomatous diseases, notably rheumatoid arthritis (where hydroxychloroquine is preferred).

PharmacokineticsChloroquine has complex pharmacokinetic properties with an enormous Vd (resulting from extensive tissue binding) and a very long elimination phase (Table 43.7). The terminal elimina-tion half-life is 1–2 months. As a consequence, the blood con-centration profile during malaria is determined mainly by distribution rather than elimination processes. Chloroquine is well absorbed by mouth. Chloroquine is approximately 55% bound to plasma proteins. The principal metabolite of chloro-quine, desethylchloroquine, has approximately equivalent anti-malarial activity. This is of relevance to prophylactic, but not therapeutic efficacy.

ToxicityChloroquine is generally well tolerated. Oral chloroquine may induce nausea or dysphoria and visual disturbances. Ortho-static hypotension may be accentuated. Pruritus is particularly troublesome in dark-skinned patients and may be dose limit-ing. Itching is described as a widespread prickling sensation mostly affecting the palms, soles and scalp which starts within 6–24 hours and may last for several days. It can be very distress-ing. Antihistamine treatment is not usually very effective. Very rarely chloroquine may cause an acute and self-limiting neuropsychiatric reaction. In prophylaxis cumulative doses over 100 g (>5 years prophylaxis) are associated with an increased risk of retinopathy. Retinal signs include a pale optic disc, arteriolar narrowing, peripheral retinal depigmentation, macular oedema, retinal granularity and oedema and retinal pigmentary changes consisting of a circle of pigmentation and central pallor; the so-called ‘doughnut’ or ‘bull’s eye’ macula.

approximately 10% at therapeutic concentrations. The effect is greater in children under 2 years of age. This can be used as a pharmacodynamic measure of toxicity. These antiarrhythmic effects are qualitatively different to the QT prolongation with quinidine, which results from delayed repolarization (JT prolongation) and can, under some circumstances, be pro-arrhythmic. Significant conduction or repolarization abnor-malities are rare and iatrogenic dysrhythmias are extremely uncommon. Quinine, like the other quinoline antimalarials, exacerbates malaria-induced orthostatic hypotension, but iat-rogenic supine hypotension is rare. Blindness, resulting from retinal ganglion cell toxicity, and deafness are common follow-ing self-poisoning, but very rare in malaria treatment. Perhaps the most important toxic effect of quinine is its stimulatory action on the pancreatic β-cell. This causes hyperinsulinaemic hypoglycaemia. It is particularly common in pregnant women but may occur in any severely ill patient, particularly if intrave-nous glucose solutions are not given. Contrary to popular opinion, quinine does not induce premature labour at thera-peutic doses. Quinine is rarely associated with a variety of aller-gic reactions, notably immune thrombocytopenia and rarely the haemolytic-uraemic syndrome. Various skin reactions are associated with quinine. Pruritus, skin flushing and urticaria are the commonest manifestations of quinine hypersensitivity. Other rashes, reported rarely, have included photosensitivity, cutaneous vasculitis, lichen planus and lichenoid photosensitiv-ity. Granulomatous hepatitis has been reported occasionally.

Blackwater fever is undoubtedly associated with quinine use, but the underlying pathophysiological mechanism is still not understood. Incorrect or non-sterile administration of intra-muscular quinine is associated with tetanus and this carries a very high mortality.

Concerns over quinine cardiovascular toxicity in severe malaria are generally exaggerated in comparison to the dangers of undertreatment and tend to arise from units in temperate countries managing occasional elderly travellers with imported malaria. Quinine is certainly potentially lethal if given by intra-venous injection, but iatrogenic hypotension is very unusual when quinine is given by rate-controlled infusion. Severe malaria is a potentially lethal condition. Antimalarial treatment is the only intervention proven to reduce mortality. Undertreat-ment may cause death – but physicians usually blame the malaria infection in a fatal case and seldom ascribe death to their use of inadequate doses of quinine. On the other hand cardiovascular complications are readily ascribed to the treat-ment rather than the fulminant disease. Large studies in endemic countries have confirmed the safety of quinine in severe malaria. It is essential that patients receiving this drug achieve therapeu-tic concentrations of quinine in their blood within hours of reaching a treatment facility.

UseParenteral quinine should be given by rate-controlled intrave-nous infusion in either 0.9% saline, 5% or 10% dextrose or by deep intramuscular injection to the anterior thigh. It should never be given by intravenous injection (as it causes potentially lethal hypotension). The initial doses (mg/kg) in children and pregnant women are the same as in non-pregnant adults, although in areas with resistant parasites it has been recom-mended that the dose be increased in children to 15 mg salt/kg from day 4 to day 8 to prevent recrudescent infection. In severe malaria, treatment should begin with a loading dose so that

576 SECTION 9 Protozoan Infections

formation of this metabolite and in theory produce a much safer compound. The incidence of these serious reactions is certainly lower when amodiaquine is used in treatment, although precise estimates of the risk are still lacking. In general artesunate-amodiaquine is well tolerated. Unusual fatigue has been prominent in some series. Case reports in the literature have documented rare neurological problems such as protrud-ing tongue, intention tremor, excess salivation and dysarthria in four African patients following amodiaquine treatment. In two patients, these signs occurred on reexposure to the drug. There is one case report of amodiaquine use over use over 1 year, resulting in yellow pigmentation of skin and mucosae, the development of corneal and conjunctival inclusion bodies and retinopathy. Minor adverse effects are similar to those of chlo-roquine, although pruritus is less of a problem and although it is still has an unpleasant taste, children find the drug more palatable.

MEFLOQUINE

Mefloquine is a fluorinated 4-quinoline methanol compound used for the treatment of multi-drug-resistant falciparum malaria. It has two asymmetric carbon atoms and is used clini-cally as a 50 : 50 racemic mixture of the erythroisomers. These have equal antimalarial activity but very different pharmacoki-netic properties. The parasiticidal action is similar to that of quinine. Mefloquine is very insoluble in water. It is available as tablets, which should be kept dry. There are no parenteral or paediatric liquid formulations. A fixed-dose co-formulation with artesunate in a 2 : 1 ratio has been developed and registered.

PharmacokineticsMefloquine is moderately well absorbed, extensively distrib-uted and slowly eliminated (Table 43.7). It is highly (>98%) bound to plasma proteins. Mefloquine is cleared principally by hepatic biotransformation to inactive metabolites. The appar-ent volume of distribution and clearance of the (+)RS enantio-mer is four to six times higher than for the (−)SR enantiomer. The overall terminal elimination half-life is approximately 3 weeks in healthy subjects and 2 weeks in malaria (Figure 43.23). The absorption of mefloquine is reduced in the acute phase of illness and bioavailability of the higher 25 mg/kg dose is improved by dividing it (e.g. giving 15 mg/kg initially and 10 mg/kg 8–24 hours later, or 8 mg/kg per day for 3 days) or in combination with artesunate delaying mefloquine administra-tion until the 2nd day of treatment. Splitting the dose also reduces the incidence of acute adverse effects. Blood concentra-tions are higher in malaria than in healthy subjects and are reduced in diarrhoea (probably by interruption of enterohe-patic recycling). Mefloquine clearance is increased in pregnancy. The pharmacokinetics in adults and children are similar. Co-administration with artesunate results in a more rapid recovery from malaria which enhances the oral bioavailability of doses after the first. Although blood concentrations are higher with split dosing early vomiting is reduced. The new fixed-dose coformulation is given as artesunate-mefloquine 4/8 mg/kg daily for 3 days.

ToxicityNausea, vomiting, dizziness, weakness, sleep disturbances and dysphoria are relatively common with mefloquine. Although

Reversible corneal opacities can be seen in 30–70% of rheuma-tology patients within a few weeks of high-dose treatment. Half are asymptomatic but others may complain of photophobia, visual halos around lights, and blurred vision. Residents on long-term chloroquine prophylaxis should probably have regular ophthalmological checks after taking the drug for 5 years, or if they experience any visual loss. Myopathy is rare at the doses used in antimalarial prophylaxis. Less common cuta-neous side effects include lightening of skin colour, various rashes (photoallergic dermatitis, exacerbation of psoriasis, bullous pemphigoid, exfoliative dermatitis, pustular rash), skin depigmentation (with long-term use) and hair loss. In self-poisoning, chloroquine produces hypotension, arrhythmias and coma and is commonly lethal. It has been suggested that diazepam is a specific antidote, but recent studies do not support a specific role for this drug above good haemodynamic and ventilatory support.

UseChloroquine is still the drug of choice for sensitive malaria parasites although ACTs are used increasingly. Chloroquine is therefore used widely for P. vivax, P. malariae and P. ovale, but except in a very few areas has been replaced for P. falciparum treatment. The time-honoured oral chloroquine regimen of 25 mg base/kg spread over 3 days (10, 10, 5 or 10, 5, 5, 5 mg/kg at 24-hour intervals) can be condensed into 36 hours of drug administration. There is no role today for parenteral chloro-quine. Chloroquine is considered safe in pregnancy and in young children.

AMODIAQUINE

Amodiaquine is a ‘Mannich base’ 4-aminoquinoline with a similar mode of action to chloroquine. It is more active against resistant isolates of P. falciparum and is combined with artesu-nate as an ACT. Amodiaquine is still effective against falciparum malaria in parts of South America, western and central Africa and a few parts of Asia, but resistance has increased. Amodia-quine is best given in the newly developed fixed-dose coformu-lation given as artesunate-amodiaquine 4/10 mg/kg daily for 3 days.

PharmacokineticsOral amodiaquine undergoes extensive first-pass metabolism by intestinal and hepatic CYP 2C8 to the biologically active metabolite desethylamodiaquine. The metabolite exerts the principal antimalarial activity. The parent compound has an elimination half-life of approximately 10 hours but desethyl-amodiaquine, like chloroquine, is extensively distributed and eliminated slowly with an estimated terminal half-life of about 10 days. There are no parenteral formulations commercially available, although a structurally similar compound, amopyra-quine, is available for intramuscular administration in some countries.

ToxicityProphylactic use of amodiaquine is associated with an unac-ceptably high incidence of serious toxicity. Approximately 1 in 2000 patients develop agranulocytosis. Serious hepatotoxicity also occurred at an estimated rate of 1 : 15 000. Agranulocytosis results from bioactivation to a reactive quinoneimine metabo-lite. Simple modifications to the chemical structure prevent

43  Malaria 577

metabolism to a biologically active desbutyl metabolite. This is eliminated more slowly (T1/2 3–7 days) than the parent com-pound and undoubtedly contributes significantly to antima-larial activity (and cardiotoxicity).

ToxicityHalofantrine is very well tolerated subjectively, but it carries a significant risk of sudden death, presumably resulting from ventricular tachyarrhythmias. Halofantrine slows atrioventricu-lar conduction and produces the ‘quinidine effect’ on myocar-dial repolarization reflected in a significant dose-related prolongation of the electrocardiograph QT interval. This is increased by previous treatment with mefloquine. This danger-ous effect is a property of both halofantrine and its desbutyl metabolite. Diarrhoea may be provoked by high halofantrine doses. There are no data for pregnancy, so halofantrine should not be used.

UseHalofantrine is only available for oral use. When used at stan-dard doses (8 mg/kg given three times at 6–8-hour intervals and repeated 1 week later in non-immune patients), in patients with a normal resting electrocardiogram, halofantrine was consid-ered safe and effective in areas with fully sensitive malaria para-sites. In multi-drug resistant areas, higher total doses are required for high cure rates, but these are associated with an unacceptable risk of cardiotoxicity. Halofantrine should not be used to treat recrudescent infections following mefloquine treatment as the cardiac effects are potentiated.

PYRIMETHAMINE

Pyrimethamine is a dihydrofolate reductase (DHFR) inhibitor. It is now used only together with long-acting sulphonamides such as sulfadoxine (as SP) and sulfalene in fixed-dose com-binations which considerably potentiate its activity. SP is not a combination, in the ‘resistance prevention’ sense. The mecha-nism of action of the two drugs is linked, so although they do provide some mutual protection, they do not protect each other from resistance to the extent unrelated drugs would. SP has been used to treat chloroquine-resistant falciparum malaria, but in many areas high-level SP resistance has devel-oped. Plasmodium vivax is also often resistant. There is an intramuscular formulation of SP but this should not be used to treat severe malaria. The DHFR inhibitors inhibit develop-ment of the mature trophozoite stage of the asexual parasite, in addition to having pre-erythrocytic and sporontocidal activities. Pyrimethamine is also used for the treatment of toxoplasmosis.

PharmacokineticsPyrimethamine is well absorbed following oral administration and is eliminated over several days (Table 43.7) (T 1/2 3 days; the companion sulfadoxine T 1/2 is 7 days), allowing single-dose treatment (Figure 43.23). Dose recommendations were devised originally in adults. A recent large study has shown that the pharmacokinetic properties of both pyrimethamine and sulfadoxine are altered significantly in children (aged 2–5 years) who have larger Vd and oral clearance values and consequently blood concentrations that are approximately half those in adults. This suggests that dose recommendations in this impor-tant age group may have been too low. Studies in pregnancy

children are more likely to vomit immediately after receiving mefloquine, and this was a significant problem when the drug was used alone, they otherwise tolerate the drug better than adults. The fixed-dose combination is associated with less early vomiting than administration of the loose tablets (possibly because of the lower mefloquine dose on day 1). Women, in particular, commonly complain of dizziness and dysphoria for up to 4 days after receiving mefloquine treatment. Mefloquine exacerbates malarial orthostatic hypotension. The main serious adverse effect of mefloquine is the development of acute but self-limiting neuropsychiatric reactions (convulsions, psycho-sis, encephalopathy). The incidence of these is approximately 1 : 10 000 when used as a prophylactic, but higher with treat-ment (1 : 1000 in Asian patients, 1 : 200 in Caucasians or Afri-cans) and 1 : 20 following severe malaria. For these reasons mefloquine should not be given following severe malaria. In one large study from Thailand mefloquine treatment in pregnancy was associated with a four-fold increased risk of stillbirth, although this effect was not seen in women exposed before conception (who would have had residual drug levels during early foetal organogenesis). This effect was not seen in the other large study experience with mefloquine in pregnancy in Malawi. The overall experience of mefloquine use over two decades sug-gests that mefloquine is safe in pregnancy.

UseMefloquine is used for the oral treatment of uncomplicated multi-drug-resistant falciparum malaria. It is given in combina-tion with artesunate 4 mg/kg per day for 3 days. The usual dose is approximately 25 mg base/kg and should be split (15 mg/kg stat. followed by 10 mg/kg 8–24 hours later, or in a fixed-dose combination at 8 mg/kg per day for 3 days). A single dose of 15 mg base/kg alone was widely used in semi-immune patients, but there is theoretical evidence that this leads more rapidly to resistance and it is no longer recommended. If the patient vomits the dose should be repeated (full dose if vomiting within 30 min, half dose 30–60 min, no further dose if after 1 hour). Mefloquine is used for antimalarial prophylaxis at a dose of approximately 4 mg base/kg once weekly for both adults and children.

HALOFANTRINE

Halofantrine is a 9-phenanthrene methanol. It has one asym-metric carbon atom and is used as a racemate. The enantiomers have equal antimalarial activity. Halofantrine is intrinsically more potent than quinine or mefloquine, but unfortunately it is associated with rare but potentially lethal ventricular tachy-cardias which have rightly curtailed its use. It is available as tablets and a suspension for paediatric use. As there are several much safer alternatives, halofantrine should be withdrawn.

PharmacokineticsHalofantrine is poorly and erratically absorbed. Furthermore, absorption appears to be ‘saturable’, i.e. with individual doses over 8 mg/kg no increment in blood concentrations occurs (Table 43.7). Absorption is increased markedly by coadminis-tration with fats. Halofantrine is extensively distributed and cleared largely by hepatic biotransformation. It is bound prin-cipally to lipoproteins in the plasma. The terminal elimination half-life is about 1–3 days in healthy subjects and approximately 4 days in patients with malaria. There is significant first-pass

578 SECTION 9 Protozoan Infections

sufficient antimalarial efficacy). This is termed ‘seasonal malaria chemoprevention’.

PROGUANIL/CHLORPROGUANIL

Proguanil (chloroguanide) and the dichlorobiguanide chlor-proguanil are considered the safest of all antimalarials. Both compounds are mainly prodrugs for the active triazine metabo-lites cycloguanil and chlorcycloguanil. The metabolites are DHFR inhibitors. The parent compounds do possess weak anti-malarial activity, probably by affecting mitochondrial electron transport.

PharmacokineticsProguanil and chlorproguanil are well absorbed by mouth reaching peak concentrations in approximately 4 hours and are converted rapidly to the triazine metabolites (Table 43.7). These in turn are metabolized to the inactive metabolites chloro- and dichlorophenylbiguanide, respectively. As the parent com-pounds are eliminated more slowly than the metabolites the profile of antimalarial activity resulting from the cyclic metabo-lites is determined by the parent drug distribution and elimina-tion. The T1/2 of proguanil has been reported as approximately 16 hours in healthy subjects and 13 hours in malaria. Recent population pharmacokinetic studies in malaria with a more sensitive assay report an estimated chlorproguanil terminal elimination half-life of 35 hours. Interestingly, the pharmaco-kinetic properties of chlorproguanil, chlorcycloguanil and dapsone are not affected by malaria. Approximately 3% of Cau-casian and African populations, but up to 20% of Orientals, fail to convert the parent compounds to their active metabolites. In some parts of Micronesia the prevalence is even higher. This is related to a genetic polymorphism in the 2C19 subfamily of the cytochrome P450 mixed-function oxidase system. The conver-sion of proguanil to the active metabolite is reduced in pregnancy.

ToxicityThe antimalarial biguanides and chlorproguanil-dapsone are very well tolerated. The biguanides occasionally cause mouth ulcers and at high doses abdominal discomfort. Hair loss has been reported. Two patients with renal failure, in whom the drugs presumably accumulated, developed pancytopenia fol-lowing prophylactic administration of proguanil. The toxicity of chlorproguanil-dapsone results from the dapsone compo-nent. The main concern is haemolysis in patients who are G6PD-deficient. Methaemoglobinaemia is also common. In large-scale evaluations chlorproguanil-dapsone caused poten-tially dangerous anaemia in African children and led to its with-drawal as an antimalarial drug. Rare idiosyncratic reactions of sulphones (like sulphonamides) include leukopenia and agran-ulocytosis, cutaneous eruptions, peripheral neuropathy, psy-chosis, toxic hepatitis, cholestatic jaundice, nephrotic syndrome, renal papillary necrosis, severe hypoalbuminaemia without proteinuria, an infectious mononucleosis-like syndrome, and minor neurological and gastrointestinal complaints.

UseProguanil has been used as a prophylactic taken once daily (3 mg/kg), often in combination with chloroquine. Chlorproguanil-dapsone has been withdrawn as a treatment of uncomplicated falciparum malaria because of haemolytic

have shown conflicting results with some studies showing lower concentrations than in non-pregnant adults. Following intra-muscular injection pyrimethamine absorption is as rapid as after oral administration but blood concentrations are lower and more variable, which suggests incomplete intramuscular bioavailability.

ToxicityPyrimethamine is very safe and well tolerated. Occasionally megaloblastic anaemia, neutropenia or thrombocytopenia may develop in patients with pre-existing folate deficiency. The tox-icity of the widely used combinations with long-acting sul-phonamides (sulfadoxine, sulfalene) or sulphones (dapsone) is almost entirely related to the sulpha components. A long list of possible adverse effects has been reported with sulphonamides. These include: (1) rare gastrointestinal toxicity: glossitis, stoma-titis, pancreatitis, salivary gland enlargement and pseudomem-branous colitis; (2) cutaneous toxicity: exfoliative dermatitis, toxic epidermal necrolysis, urticaria, photosensitivity, cutane-ous vasculitis, erythema nodosum, lichen planus, pruritus and hair loss; (3) CNS: dizziness, ataxia, benign intracranial hyper-tension, aseptic meningitis, hearing loss, tinnitus, reversible peripheral neuropathy; (4) renal effects: proteinuria, haematu-ria, acute interstitial nephritis, crystalluria (older sulphas – not generally associated with sulfadoxine); (5) haematological; thrombocytopenia, antibody-mediated haemolysis, neutrope-nia and (6) drug fever. The sulphones commonly cause meth-aemoglobinaemia, cause haemolytic anaemia in G6PD deficiency and also rarely cause blood dyscrasias.31 Severe reac-tions occurred in about 1 : 7000 subjects receiving sulfadoxine-pyrimethamine prophylaxis (mortality was 1 : 18 000). The risk with treatment use is almost certainly much lower, but there are no precise estimates.

UseCombinations of pyrimethamine with long-acting sulphon-amides (SP) should not be used for prophylaxis, whereas the sulphone combination, which appears to be safer (at a once-weekly dose), is used occasionally for prophylaxis but not treat-ment. Pyrimethamine alone is no longer prescribed as an antimalarial. SP is given in a single oral dose ensuring that this contains a minimum of 1.25 mg/kg of pyrimethamine. This should be combined with a 3-day course of artesunate (4 mg/kg per day) to improve treatment efficacy. This is well tolerated, inexpensive and effective where efficacy of SP remains high (cure rates >80%). SP is regarded as safe in pregnancy. Studies from Africa indicate that administration of SP at least three times during pregnancy (at intervals ≥1 month) has a beneficial effect on maternal anaemia and pregnancy outcome (birth weight). HIV-positive women need SP more frequently for the same effect. This approach has now been extended to infancy and possible incorporation in EPI programmes. Whether other drugs are as good as SP in IPT, whether the findings in high transmission settings apply also to low transmission settings, whether artesunate should be added and what to do when resis-tance develops, all remain to be determined. Across the Sahel where malaria transmission is intense over 3–4 months each year it is now recommended that monthly treatment courses of amodiaquine and SP should be given to children aged between 3 and 59 months at monthly intervals, beginning at the start of the transmission season, to a maximum of 4 doses during the malaria transmission season (provided both drugs retain

43  Malaria 579

PRIMAQUINE

Primaquine is an 8-aminoquinoline used mainly for its actions against the hypnozoites of P. vivax (to prevent relapse) and the gametocytes of P. falciparum (to prevent transmission). Prima-quine has significant liver stage pre-erythrocytic activity against all the malarias (which accounts for its prophylactic efficacy) and it also has significant activity against asexual stage parasites of P. vivax, P. malariae and P. ovale. Thus, the radical treatment of vivax and ovale in infections, where primaquine is combined usually with chloroquine, is a combination treatment which should provide mutual protection against resistance.

PharmacokineticsPrimaquine is well absorbed after oral administration. It is cleared by hepatic biotransformation with an elimination half-life of 8 hours to the more polar biologically inactive metabolite carboxyprimaquine, which is eliminated more slowly. Metabo-lism via cytochrome P450 biotransformation produces several other metabolites. It is not clear which of these highly reactive metabolites mediates the biological effects of primaquine.

ToxicityNausea, headache, vomiting and abdominal pain or cramps are relatively common particularly if higher doses of primaquine (>30 mg) are taken on an empty stomach. Taking primaquine with food considerably improves tolerability. At an adult dose of 15 mg mild abdominal pain was reported in 3% of US servicemen and 22.5 mg produced abdominal symptoms in 12% which required treatment in 3%. In general adult doses of 30 mg base are well tolerated if taken with food. Mild diarrhoea, chest pain, weakness, visual disturbances and pruritus occur occasionally. Significant methaemoglobinaemia (>10%) such that the patient appears cyanosed occurs in less than 10% of adult patients receiving ≤22.5 mg/day. The principal toxicity of primaquine is oxidant haemolysis. This may result from oxidant species induced by the phenolic metabolite 5-hydroxyprimaquine. This is the most serious side-effect in individuals with glucose 6 phosphate dehydrogenase (G6PD) deficiency, other enzyme deficiencies (e.g. glutathione synthe-tase) that counter oxidant stress, and several haemoglobinopa-thies (e.g. Hb Zurich, Hb Torino). Although haemolytic toxicity first recognized in the 1920s with plasmoquine, it was not until the early 1950s that the sex-linked G6PD deficiency was discov-ered. The severity of haemolysis is related to the degree of G6PD deficiency and the primaquine dose. Haemolysis is, therefore, generally less severe in the African (A-) form. In such patients, haemolysis tends to be self-limiting but in some of the Asian variants and the Mediterranean type haemolysis may be severe. There are a large number of different G6PD genotypes and there is considerable phenotypic variation within the geno-types. This makes generalizations about the risks of haemolysis difficult. Essentially all subjects who are G6PD deficient (<30% of normal activity) will haemolyse following primaquine administration to an extent determined by the degree of defi-ciency and the dose and duration of treatment. Fortunately because primaquine is eliminated rapidly haemolysis is self-limiting if the drug is stopped. There is insufficient information on the relationship between genotype, red cell G6PD concen-trations and haemolytic tendency with primaquine. Haemolysis may be exacerbated by concurrent infections, liver disease (altered primaquine metabolism), renal impairment (delayed

toxicity. The treatment doses of proguanil used are 5–8 mg/kg per day (in combination with atovaquone).

ATOVAQUONE-PROGUANIL

This highly active hydroxynaphthaquinone antimalarial drug is active even against multi-drug-resistant falciparum malaria. The speed of therapeutic response is similar to that with meflo-quine and slower than that with artemisinin derivatives. Origi-nally atovaquone alone was developed, but high-level resistance developed in approximately 30% of treated patients. This sug-gested that the point mutations in cyt b, which conferred resis-tance, occurred at an approximate frequency of 1 in 1012 parasites. The fixed combination with proguanil proved much more effective producing cure rates of nearly 100% and emer-gence of resistance in less than 1 : 500 treated patients. It is this combined formulation (Malarone®), which is registered both for prophylaxis and treatment use in many countries. Neverthe-less it must be considered vulnerable and for treatment use in endemic areas should ideally be combined with an artemisinin derivative. This creates a highly effective and well-tolerated arte-misinin based combination treatment. Interestingly it is the parent compound proguanil which is the important contribu-tor to antimalarial efficacy, as atovaquone-proguanil is equally effective against highly antifol-resistant parasites and also in individuals unable to convert proguanil to cycloguanil. Unfor-tunately, the very high cost of atovaquone synthesis makes this drug largely unaffordable in tropical countries.

PharmacokineticsAtovaquone is similar to halofantrine and lumefantrine in that oral absorption is augmented considerably by fats (Table 43.7). Elimination is slower in patients of African origin (T1/2 70 hours) than in Oriental patients (T1/2 30 hours). There are no significant interactions with proguanil or artesunate. Con-centrations of both components are reduced by almost one half in late pregnancy Atovaquone is eliminate.

ToxicityThe combination is really very well tolerated. Atovaquone-proguanil may cause vomiting in some patients. The adverse effects otherwise are similar to those of proguanil.

UseAtovaquone-proguanil is becoming established as a safe, effec-tive and expensive antimalarial prophylactic for travellers – as it is effective everywhere. The adult prophylactic dose is one tablet (atovaquone 250 mg proguanil 100 mg) daily with food. The treatment dose is 15–20/6–8 mg/kg per day for 3 days, which corresponds to an adult dose of 4 tablets per day. It is equally efficacious and well tolerated in young children. The triple combination with artesunate is well tolerated and highly effective against multidrug-resistant falciparum malaria and should be given if this drug is used in endemic areas. Artesunate-atovaquone-proguanil has been evaluated in pregnant women failing other treatments. It was well tolerated and effective, although plasma levels of all components were reduced suggest-ing that a higher dose would be needed. For treatment use cost has been a major barrier to its use and its use is almost exclu-sively confined to the treatment of imported malaria in temper-ate countries and in very limited deployments against artemisinin-resistant falciparum malaria in Eastern Thailand.

580 SECTION 9 Protozoan Infections

converted back to it within the body (Figure 43.26). Artemisinin itself is available in a few countries. It is 5–10 times less active than the derivatives and is not metabolized to DHA. These drugs are the most rapidly acting of known antimalarials and they have the broadest time window of antimalarial effect (from ring forms to early schizonts). They produce more rapid para-site clearance than other antimalarial drugs and they have proved to be very safe in clinical practice. They are simply the best drugs for severe malaria (Figure 43.27). In a large random-ized controlled trial in severe malaria, conducted in four Asian countries, artesunate reduced the mortality of severe malaria by 35% compared with quinine. Most of these patients were adults (n = 1461; of whom 202 were children). In a subsequent trial, the largest ever conducted in severe malaria, conducted in 5425 African children, artesunate reduced mortality by 22.5% (Figure 43.28).32 There were no serious adverse effects. Artesu-nate reduced the incidence of hypoglycaemia, convulsions and neurological deterioration and importantly did not increase the incidence of neurological sequeale. These are very large mortal-ity reductions and they have established artesunate as the treat-ment of choice for severe malaria. Later investigations based on estimation of the parasite biomass using plasma PfHRP2 sug-gested that some of this difference between the mortality reduc-tion in Asia and that in Africa (35% vs 22.5%) might be explained by overdiagnosis of severe malaria in African chil-dren, i.e. that some children fulfilling the definition of severe malaria die from another pathological process – most likely sepsis.13 In falciparum malaria the artemisinin derivatives also effectively prevent progression to severe disease. For example in western Thailand a parasitaemia over 4% without vital organ dysfunction carries a 3% mortality (i.e. 30 times higher than uncomplicated malaria but less than one fifth that of severe

Figure 43.26 Artemisinin combination treatment. The effects of adding a 3-day course of artesunate to high-dose (25 mg/kg) meflo-quine on malaria parasite killing in an area of pre-existing mefloquine resistance (e.g. in Thailand). The parasite burden is relatively high in the example (corresponding to about 2% parasitaemia). Without the arte-sunate the parasitaemia declines 100-fold per asexual cycle and is elimi-nated finally in 3 weeks. If artesunate is added for 3 days, covering two asexual cycles, then the parasite biomass is reduced 108-fold leaving a maximum residuum of only 105 parasites remaining (and usually much fewer) for the high concentrations of mefloquine to remove (B). This offers a hundred-million-fold lower opportunity of selecting a resistant parasite. Note that without artesunate the corresponding number of parasites (B1) ‘see’ much lower concentrations of mefloquine (from x to y, compared with m to n) and have therefore an increased risk of recrudescing.

1012

102

106

108

1010

104Tota

l par

asite

s

0

Weeks

Detection limitDrug level

A

B B1

m

n

x

y

0 1 2 3 4

excretion) and co-administration of other drugs with haemo-lytic potential, e.g. sulphonamides. Primaquine is contraindi-cated in pregnancy.

UseRadical curative activity depends on the total dose administered and this is limited by adverse effects. Primaquine is given once daily in a dose of 0.25–0.5 mg base/kg (adult doses 15–30 mg) together with food. The higher dose is now recommended for tropical ‘strains’ and the usual course of treatment for the radical treatment of vivax and ovale malaria is 14 days. Shorter courses with higher doses have also proved effective (30 mg twice daily for 7 days). In particular, there is no evidence that the previously recommended 0.25 mg base/kg per day for 5-days is effective. There is no evidence for adverse interactions with other antimalarial drugs but this has been little studied. In patients with mild G6PD deficiency a once weekly dose of 0.6–0.8 mg/kg (adult dose 45 mg) is given for 8 weeks. For P. falciparum gametocytocidal activity a single dose of 0.5–0.75 mg base/kg is recommended although recent evidence suggests that a single 0.25 mg base/kg dose gives maximal transmission blocking activity with less haemolysis in G6PD-deficient patients. For prophylaxis the adult dose evaluated has been 30 mg daily taken with food. This has been remarkably well tolerated. In most vivax-endemic areas G6PD deficiency is prevalent but testing is not available and there is no consensus on how primaquine should be used in these circumstances. If significant haemolysis occurs primaquine should be stopped and the patient observed. Transfusion is rarely necessary except when there is severe deficiency.

TAFENOQUINE

Formerly known as etaquine or WR 238605, this slowly elimi-nated 8-aminoquinoline was developed by the US army. It is currently undergoing phase III trials in antimalarial prophylaxis and for the radical treatment of vivax malaria. Tafenoquine has a terminal elimination half-life of approximately 2 weeks. Tafenoquine is more efficacious and better tolerated than pri-maquine, although it still causes oxidant haemolysis in G6PD-deficient subjects.

METHYLENE BLUE

Methylene blue was first shown to have antimalarial activity by Gutthman and Ehrlich over 100 years ago. Recently interest has been rekindled in this well-established dye and studies com-pleted showing antimalarial activity in vivo and gametocytoci-dal activity against P. falciparum in vitro.

QINGHAOSU (ARTEMISININ)

Qinghaosu or artemisinin is a sesquiterpene lactone peroxide extracted from the leaves of the shrub Artemesia annua (Qinghao). Four derivatives are used widely: the oil-soluble methyl ether artemether (or the very similar compound arte-motil formerly known as arteether), the water-soluble hemisuc-cinate derivative artesunate and dihydroartemisinin (DHA). A semisynthetic derivative artemisone and fully synthetic trioxa-lone compounds (OZ 277; arterolane and OZ 439) with similar modes of action are under development. Artesunate, artemether and artemotil are all synthesized from DHA and they are

43  Malaria 581

Figure 43.27 Qinghaosu: the parent compound artemisinin and the three derivatives. The oil-soluble ethers, artemether and artemotil (arteether) and the water-soluble artesunate are all converted in vivo to a common biologically active metabolite dihydroartemisinin. The per-oxide bridge in the sesquiterpene structure is essential for antimalarial activity.

Qinghaosu(Artemisinin)

Dihydroqinghaosu(Dihydroartemisinin)

CH3

CH3

CH3H

HH

O

O

O

OO

CH3

CH3

CH3H

HH

OH

O

O

OO

Artemether

CH3

CH3

H

O

O

Arteether

CH3

C2H5

H

O

O

Artesunate

CH3

C

H

O CH2CH2COOH

O

O

Figure 43.28 Forest plots from individual patient data analyses of randomized trials comparing parenteral artesunate versus the standard quinine loading dose regimen in severe falciparum malaria. These show a highly significant difference between artesunate and quinine. Artesunate reduced mortality by 35% in adults and by 22.5% in children.

Artesunate

230/2712 (8.5%)

1/33 (3%)

107/730 (14.7%)

7/59 (11.9%)

4/37 (10.8%)

5/31 (16.1%)

2/24 (8.3%)

Quinine

297/2713 (10.9%)

2/33 (6.1%)

164/731 (22.4%)

12/54 (22.2%)

5/35 (14.3%)

8/30 (26.7%)

23/67 (34.3%)

Study

Africa

AQUAMAT 2010

Sudan 2010

Subtotal (X2=0.06, df=1, p=0.81)

Asia

SEAQUAMAT 2005

Thailand 2003

Vietnam 1997

Vietnam 1992

Myanmar 1992

Subtotal (x2=1.80, df=4, p=0.77)

Overall (x2=4.51, df=6, p=0.61)

Heterogeneity between continents: x2=2.65, df=1, p=0.10

OR (95% Cl) P(99% Cl for totals)

0.75 (0.83, 0.90) 0.002

0.48 (0.01, 9.85) 0.56

0.75 (0.59, 0.95) 0.002

0.60 (0.45, 0.79) 0.0002

0.47 (0.14, 1.44) 0.14

0.73 (0.13, 3.75) 0.66

0.53 (0.12, 2.17) 0.32

0.17 (0.02, 0.83) 0.02

0.58 (0.41, 0.81) 0.00005

0.69 (0.57, 0.84) <0.000001

Favours artesunate

Overall or 0.69 (0.57 to 0.84), p<0.000001

Favours quinine

0.1 0.3 0.7 1 2 3 4

malaria). In this context, oral artesunate produces considerably superior therapeutic responses compared with an intravenous quinine loading dose. This property of rapidly stopping parasite development and thereby arresting progression of the infection also prevents development of severe malaria. Before the recent definitive studies of artesunate trials in severe malaria were conducted mainly with artemether. In randomized controlled trials which together enrolled nearly 2000 patients, intramus-cular artemether was associated with a significantly lower mor-tality in South-East Asian adults when compared with quinine, but there was no significant difference in African children. Arte-mether was not associated with more rapid clinical responses (fever clearance, coma recovery) but it did accelerate parasite clearance. But artemether (or artemotil) were not the best drugs to have chosen. Although they are highly effective in vitro, in vivo these oil-based intramuscular injections were found to be slowly and erratically absorbed – particularly in the most severely ill patients. This pharmacokinetic disadvantage coun-tered the intrinsic pharmacodynamic advantage of this drug class. The water-soluble artesunate by contrast is reliably and rapidly absorbed following intramuscular injection, and can be given intravenously. In a recent large randomized comparison of artesunate versus artemether conducted in Vietnam there was a borderline mortality advantage to artesunate. Thus arte-sunate is better than artemether, which is in turn better than quinine in the treatment of severe malaria.

Most deaths from severe malaria take place in or near home and far from facilities capable of providing injections. Rectal formulations of artemisinin and artesunate have been devel-oped for community use as treatments for patients suspected of having severe malaria, who are febrile and unable to take

582 SECTION 9 Protozoan Infections

Weight (kg) Age Artesunate Dose (mg) Regimen (Single Dose)

5–8.9 0–12 months 50 One 50-mg suppository9–19 13–42 months 100 One 100-mg suppository

20–29 43–60 months 200 One 100-mg + one 50-mg suppository30–39 6–13 years 300 Three 100-mg suppositories>40 >14 years 400 One 400-mg suppository

TABLE 43.8

Dosage for Rectal (Rectocap) Artesunate Initial (Pre-Referral) Treatment in Children (Aged 2–15 Years) and Weighing at Least 5 kg

medications by mouth. A rectal formulation of artesunate was evaluated in a very large multicentre trial, conducted in Ghana, Tanzania and Bangladesh (Table 43.8). Preferral administration of rectal artesunate reduced the mortality of children under 5 years with malaria who could not tolerate oral treatment by 25%.33

In clinical studies in uncomplicated falciparum malaria the artemisinin derivatives provide both more rapid parasite and fever clearance than with other treatments and also reduce gametocyte carriage and thus transmission. Concerns over their neurotoxic potential, revealed in animal studies, have not been confirmed in large and detailed clinical, neurophysiological and pathological studies. Indeed their remarkable safety, efficacy and lack of adverse effects have led to widespread unregulated use and the manufacture of fake products.

Artemisinin is available as capsules of powder or as supposi-tories. Artemether is formulated in peanut oil and arteether in sesame seed oil, for intramuscular injection and in capsules or tablets for oral use. Artesunate is formulated either as tablets, in a gel enclosed in gelatin for rectal administration (called a Rectocap™), or as dry powder of artesunic acid for injection, usually supplied with an ampoule of 5% sodium bicarbonate. The powder is dissolved in the sodium bicarbonate, to form sodium artesunate and then diluted in 5% dextrose or ‘normal’ saline for intravenous or intramuscular injection. The majority of clinical data pertain to the most widely used derivative artesunate.

The recent emergence of resistance to these drugs in South-east Asia is of major concern, threatening their life-saving benefit in severe malaria and their remarkable efficacy in uncomplicated malaria. For the vast majority of the malaria-affected world ACTs are highly effective. The rapid therapeutic responses prevent the development of severe malaria and allow earlier return to school or work, mutual protection against resistance and reduced gametocyte carriage which may reduce the incidence of malaria in low transmission settings. Com-munity use of these drugs as monotherapies is strongly discour-aged by the World Health Organization to reduce selective pressure for resistance.

PharmacokineticsThe artemisinin derivatives are rapidly absorbed and eliminated (Table 43.7). Artemisinin is cleared by metabolic conversion to inactive metabolites. It is a potent inducer of its own metabo-lism. Artesunate, artemether and artemotil are converted to the active metabolite dihydroartemisinin, which has an elimination half-life of approximately 45 min. Parenteral artesunate is hydrolysed rapidly at neutral pH and this is accelerated by plasma esterases. Artemether and artemotil are converted by hepatic biotransformation. Although they are by far the most rapidly eliminated of the antimalarial drugs, because of their

broad stage specificity of action they are highly effective when given once daily. Unlike some antibiotics it is not necessary to exceed the MIC throughout the dosing interval for antimalarial drugs (after all it is only necessary to kill each parasite once!). After oral or parenteral administration artesunate is hydrolysed rapidly (by stomach acid and esterases in plasma and erythro-cytes) and most of the antimalarial activity results from the DHA metabolite. Oral absorption is rapid and bioavailability is approximately 60%. Rectal bioavailability is more variable; fol-lowing administration of the Rectocap™ bioavailability averages 50% (although rates of absorption vary widely). As for quinine there is a contraction in the volume of distribution and reduced clearance in acute malaria, which increases blood concentra-tions. There is also be a malaria-related inhibition of intestinal and hepatic first pass metabolism (by glucuronidation) which improves oral bioavailability. After oral administration arte-mether is absorbed rapidly, but is converted more slowly (via CYP 3A4) to DHA, although the metabolite still accounts for the majority of antimalarial activity. In contrast after intramus-cular administration absorption of artemether and artemotil (arteether) is slow and erratic. Peak concentrations are often not reached for many hours. Following intramuscular administra-tion of artemether concentrations of the parent compound exceed those of the active DHA metabolite. In some patients with severe malaria absorption may be inadequate. Oral formu-lations of DHA contain excipients which promote absorption and give bioavailability comparable to that of artesunate. Elimi-nation of DHA is largely by conversion to inactive glucuronides. No significant drug interactions other than autoinduction (artemisinin>artemether>DHA) have been identified with these compounds. Concentrations of artemisinin derivatives and DHA are similar in children and adults.

ToxicityThe artemisinin-related compounds have been remarkably well tolerated in clinical evaluations. There has been no documented significant toxicity other than rare type 1 hypersensitivity reac-tions (incidence approximately 1 : 3000 treatments). In volun-teer studies a depression of reticulocyte counts has been noted and haemoglobin recovery in the 1st week is slightly slower than with other antimalarials but increased anaemia thereafter has not been observed in clinical studies. When given at high doses (≥6 mg/kg per day) over several days artesunate causes self-limiting neutropenia. Following treatment of hyperparasitae-mia a haemolytic anaemia may be seen resulting from shortened survival of once-parasitized red cells. In animal studies the arte-misinin derivatives are much less toxic than the quinoline anti-malarials. The principal toxicity in animals has been an unusual dose-related selective pattern of neuronal cell damage affecting certain brain stem nuclei. This is related to the pharmacokinetic properties of the drug. Neurotoxicity is related to protracted

43  Malaria 583

undertaken. These drugs are still not recommended for the treatment of uncomplicated falciparum malaria in early preg-nancy (1st trimester), unless there are no effective alternatives, but this may change. There is now confidence in their safety in the 2nd and 3rd trimesters and ACTs should be used – although higher dosages or longer courses may be needed for optimum efficacy. Thus overall no adverse effects on the pregnancy or infant development have been seen in prospective studies.

UseIn severe malaria, artesunate is given by intravenous or intra-muscular injection. The doses are 2.4 mg/kg given at 0, 12, 24 hours, then daily.34 Artemether and artemotil (arteether) are given by intramuscular injection to the anterior thigh. The dose of artemether is 3.2 mg/kg initially followed by 1.6 mg/kg daily (Table 43.9). The artesunate Rectocap is used in a dose of 10 mg/kg per day until parenteral or oral treatment can be given (Table 43.8). For oral treatment fixed-dose ACTs are preferred (Table 43.10). If used alone, the artemisinin derivatives should be given in a 7 (not 5)-day course, but this should be combined with doxycycline or clindamycin where possible. The initial oral

exposure related to sustained blood concentrations, as follows intramuscular administration of the oil-based artemether and arteether. Much less neurotoxicity is seen following oral admin-istration or intravenous artesunate because the drug levels are not sustained. Extensive clinical neurophysiological and, to a lesser extent, pathology studies have failed to show any evidence of neurotoxicity or cardiotoxicity in clinical use. Initial animal studies also suggested effects on the electrocardiographic QT interval (ventricular repolarization) but this was probably sec-ondary to neurotoxicity. These drugs do not affect the heart in clinical use. The main concern over their use relates to early pregnancy. In experimental animals exposure during a critical time window in early pregnancy causes fetal loss as a result of inhibition of erythropoiesis. Whether this effect could produce fetal developmental abnormalities in primates and therefore could be teratogenic in clinical use in the treatment of malaria was of great concern. Prospective clinical studies in over 1000 exposed pregnancies, including over 100 1st trimester expo-sures is reassuring to date.26 Indeed, given their superior efficacy over other drugs and the harmful effects of malaria illness com-parative trials with ACTs in the 1st trimester are being

Hospital Health Clinic Rural Health Clinic

Intensive care unit (ICU) No intravenous infusions possible No injection facilitiesArtesunate 2.4 mg/kg stat. by IV injection

followed by 2.4 mg/kg at 12, and 24 hours then daily if necessary

As for hospital ICU: artesunate can also be given by IM injection

Artesunate Rectocap: 10 mg/kg daily

Artemisinin suppository 20 mg/kg at 0 and 4 hours, then daily

IF ARTESUNATE UNAVAILABLEArtemether 3.2 mg/kg stat. by IM injection

followed by 1.6 mg/kg daily

IF ARTESUNATE AND ARTEMETHER UNAVAILABLEQuinine dihydrochloride 20 mg salt/kg infused

over 4 hours.Maintenance dose: 10 mg salt/kg infused over

2–8 hours at 8-hour intervals

Quinine dihydrochloride 20 mg salt/kg diluted 1 : 2 with sterile water given by split injection into both anterior thighs.

Maintenance dose: 10 mg/kg 8-hourly

General points: Infusions can be given in 0.9% saline, 5% or 10% dextrose/water. Infusion rates for quinine should be carefully controlled. Oral treatment should start as soon as the patient can swallow reliably enough to complete a full course of treatment. A full course of artemisinin combination treatment should be given.

TABLE 43.9 Antimalarial Drug Doses in Severe Malaria

ARTEMETHER-LUMEFANTRINEA

Age (years) Body weight (kg) Number of Artemether-lumefantrine tablets per dose (twice daily for 3 days)<3 5–14 13–9 15–24 29–14 25–34 3>14 >34 4

ARTESUNATE-AMODIAQUINE, ARTESUNATE-SP, ARTESUNATE-MEFLOQUINEAge (years) Number of Artesunate

50 mg tablets per dose (Daily for 3 days)

Number of Amodiaquine 153 mg tablets per dose (Daily for 3 days)

Number of Sulfadoxine-Pyrimethamine 25/500 mg tablets (single dose)

Number of Mefloquine 250 mg tablets

(Daily for 3 days)

½ to 1 ½ ½ ½ ¼1–6 1 1 1 ½7–13 2 2 2 1>13 4 4 3 2

These are dosage guides, but adjusting tablets on the basis of weight is preferable.aThe drug should be taken with food or milk.

TABLE 43.10 Oral ACT Dosing Schedules

584 SECTION 9 Protozoan Infections

dose is 4 mg/kg followed by 2 mg/kg per day. Dose restrictions are not necessary in renal failure or with liver disease. The arte-misinin derivatives are safe in infants and children. Until more information is available as a policy of caution they should not be used in the 1st trimester of pregnancy (although there is no evidence of harm), but should be used in the 2nd and 3rd tri-mesters. These drugs should be used for the treatment of severe malaria in pregnancy at any gestational age as they save lives and they are safer than quinine.

ARTEMISININ COMBINATION TREATMENTS

The ACTs are rapidly effective and generally reliable treatments. The artemisinin derivatives induce a rapid resolution of fever and illness. This may improve absorption of the combination partner (mefloquine, lumefantrine). While present in the blood (usually 3 days) they also protect the partner drug from the emergence of resistance and they reduce gametocyte carriage. The partner drug then removes the relatively few parasites remaining after the 3-day course of treatment (a hundred million times less than when treatment started) and also pro-tects the artemisinin derivative from resistance (Figure 43.27). But once the artemisinin derivative has been eliminated the partner compound is no longer ‘protected’ and may then select for resistance. Thus, provided the partner is effective and full doses are absorbed protection of the artemisinin component from resistance is complete, whereas protection of the partner drug is incomplete. WHO currently recommends five ACTs; artesunate-amodiaquine, artesunate-sulphadoxine–pyrimethamine (in areas where prevalent malaria parasites are a sensitive to the partner drugs) and artesunate-mefloquine, artemether-lumefantrine and dihydroartemisinin-piperaquine which can be used everywhere.34

ARTEMETHER-LUMEFANTRINE

Formerly called benflumetol, lumefantrine was developed by Chinese scientists. It is now available only in a fixed tablet com-bination with artemether. Each tablet contains artemether 20 mg and lumefantrine 120 mg. This is the most widely used ACT in the world accounting for approximately 70% of all use. Artemether-lumefantrine is very effective against multi-drug-resistant falciparum malaria and it is remarkably well tolerated.

PharmacokineticsLumefantrine is lipophilic and hydrophobic (Table 43.7). Its absorption is dose-limited and is considerably augmented by taking the drug together with food (a 16-fold increase with a fatty meal). Only a small amount of fat is required. Dose- finding studies with soya milk showed 36 mL (equivalent to 1.2 g fat) were required to produce 90% of maximum absorp-tion. The absorption of lumefantrine is reduced in the acute phase of malaria, but then increases considerably as symptoms resolve and the patient starts to eat. Absorption is capacity limited so increasing the current dose does not provide a cor-responding increase in the amount of drug absorbed. This means the drug cannot be given once daily. Lumefantrine is metabolized to a desbutyl metabolite (principally by CYP 3A4) which has greater antimalarial activity but contributes relatively little to overall antimalarial effect. The elimination half-life is 3–4 days. As a result it provides a shorter duration of

post-treatment prophylaxis compared with more slowly elimi-nated drugs such as mefloquine and piperaquine. This results in earlier recurrences and less suppression of P. vivax relapses. The pharmacokinetic properties of lumefantrine are similar in adults and children. The principal pharmacokinetic variable which correlates with therapeutic response is the area under the plasma concentration-time curve (AUC). The plasma or whole blood level on day 7 after starting treatment is a good surrogate of the AUC for this and other slowly eliminated antimalarials. Plasma concentrations of both drug components are reduced by approximately half in pregnancy so the current dose regimen is insufficient for optimum cure rates in this important patient group.

ToxicityThis combination is remarkably free of adverse effects. Con-cerns about possible cardiotoxicity have been refuted by careful studies. Lumefantrine is not cardiotoxic.

UseThere is now extensive experience with artemether-lumefantrine from all parts of the malaria-affected world attesting to safety and efficacy. The treatment course initially recommended was 1.5/ 9 mg/kg (adult dose 4 tablets) given at 0, 8, 24 and 48 hours. This was effective in patients with background immunity, but cure rates in non-immune patients with multi-drug-resistant infections were approximately 80%. Increasing the regimen to 6 doses (i.e. twice daily on each day) resulted in >95% cure rates and this regimen has proved effective across the world. Where it has been assessed, adherence to this regimen has been rela-tively good. The patient should be encouraged to take the drug with food or a small amount of milk. Several studies suggest artemether-lumefantrine is safe in the 2nd and 3rd trimesters of pregnancy, although more information is needed and dose optimization is required. A paediatric formulation has been developed.

PYRONARIDINE

Structurally a relative of amodiaquine, pyronaridine has been developed and used in China. It is active against multidrug-resistant Plasmodium falciparum malaria35 and, like many drugs in this class, it is extensively distributed and slowly eliminated. Originally pyronaridine was deployed as an enteric-coated for-mulation for monotherapy (which had poor oral bioavailabil-ity) and was given in a 3-day course of 1200 mg or 1800 mg (adult dose) over 5 days. Pyronaridine has been developed as the tetraphosphate and now registered as a fixed co-formulation with artesunate in a 3 : 1 dose ratio. The efficacy of artesunate-pyronaridine against all malaria tested to date has been excellent.

PharmacokineticsThere are few data on the pharmacokinetics of pyronaridine in the public domain. In phase II clinical trials on the pharmaco-kinetics, clinical and safety outcome of artesunate-pyronaridine carried out in 16 patients in Uganda with acute falciparum malaria indicated a large apparent volume of distribution and a long elimination phase. The latest assessment of the terminal elimination half-life of pyronaridine is approximately 10–13 days. Pyronaridine is concentrated in red blood cells.

43  Malaria 585

falciparum and vivax malaria. When measured adherence has been excellent. Recent studies also indicate good efficacy and excellent tolerability in African children. The long period of post-treatment prophylaxis conferred by the slowly eliminated piperaquine is both an advantage in preventing reinfections and suppressing P. vivax relapses, but also increases the selection pressure on resistance.

ANTIBACTERIALS WITH ANTIMALARIAL ACTIVITY

The antibacterials which act on protein or nucleic acid syn-thesis often have significant antimalarial activity. But they are not sufficiently active to be used alone to treat malaria. The sulphonamides and sulphones inhibit plasmodial folate syn-thesis by competing for the enzyme dihydropteroate synthetase. The sulphonamides and sulphones are usually used in com-bination with pyrimethamine or the antimalarial biguanides with which they are synergistic. Trimethoprim is also an antifol; it has good antimalarial activity and shares resistance profiles with pyrimethamine. The tetracyclines are consistently active against all species of malaria. Doxycycline is the most widely used both for prophylaxis and treatment. Clindamycin is as effective as the tetracyclines and has the advantage that it can be used in children and pregnant women. The macrolides are active in vitro but are generally disappointing in vivo. Azithro-mycin is more active and has been evaluated both in prophy-laxis and treatment. Rifampicin has a weak antimalarial effect in vivo. Chloramphenicol has antimalarial activity but this has not been well characterized. The fluoroquinolones have some activity but, despite one promising sentinel report, sub-sequent clinical experience has proved uniformly disappoint-ing. Fosmidomycin has good antimalarial activity and is under investigation. These drugs all act relatively slowly and they are therefore used in combination with more rapidly acting agents.

ANTIMALARIAL DRUG INTERACTIONS

Antimalarial drug interactions have not been well character-ized. Mefloquine, halofantrine, quinidine and quinine are struc-turally similar and may compete for blood and tissue binding sites. Cardiotoxicity is assumed to be additive and significant only for halofantrine, where there is a potentially dangerous interaction with mefloquine. It has been recommended that mefloquine should not be given to people also receiving quinine to avoid adverse cardiovascular effects, but no interaction has been demonstrated. Inducers of CYP 3A4 such as rifampicin, ritonavir boosted lopinavir and anticonvulsant drugs accelerate the clearance of quinine and mefloquine resulting in lower drug levels (and a greater chance of treatment failure). There is no evidence that the structurally dissimilar antimalarials interact. Use of artesunate together with mefloquine improves the tolerance and absorption of mefloquine presumably by curing malaria more rapidly. Similarly, the absorption of lume-fantrine improves as the patient recovers. There are several potential or proven interactions with antiretroviral drugs usually resulting in reduced antimalarial exposure. For example nevirapine reduces oral artemether and DHA exposure. On the other hand protease inhibitors may increase quinine exposure. Amodiaquine has a toxic interaction with efavirenz resulting in hepatotoxicity.

ToxicityAlthough generally well tolerated, there remains some concern over the potential of pyronaridine for hepatotoxicity. This is being addressed in Phase IV trials.

UseArtesunate-pyronaridine is highly effective against all malarias, including multi-drug-resistant falciparum malaria.

PIPERAQUINE

Also developed in China this bisquinoline compound and its hydroxy-derivative are active against multidrug-resistant Plasmodium falciparum.36 Piperaquine replaced chloroquine in China as first-line treatment for falciparum malaria in 1978 and was used until 1994. Over 200 tonnes were dispensed. Resis-tance reportedly developed, but reversed after piperaquine was discontinued. In recent years, piperaquine has been developed as a fixed combination with dihydroartemisinin. The currently available formulation contains 40 mg of dihydroartemisinin and 320 mg of piperaquine (as the phosphate) per tablet and is given in an adult dose of 3 tablets/day (equivalent to approx. 2.3/16 mg/kg, once daily for 3 days. It is relatively inexpensive (adult doses currently just over US$1). This combination is registered, endorsed by the World Health Organization and used increasingly. The combination is well tolerated and effec-tive everywhere.

Another combination artemisinin-piperaquine (as the base) is also available in some areas.

PharmacokineticsOral dihydroartemisinin absorption is very dependent on the formulation and excipients. In current formulations, it is reli-ably and rapidly absorbed. Piperaquine is more slowly absorbed. It is extensively distributed and very slowly eliminated. The pharmacokinetic properties are similar to those of chloroquine. Absorption is slightly increased by fats. Latest estimates for the terminal elimination half-life are approximately 1 month. Young children have lower plasma concentrations than adults, particularly 1–2 weeks after drug administration and therefore need a higher mg/kg dose than adults. As with other slowly eliminated antimalarials the day 7 plasma or blood concentra-tion is a valuable predictor of efficacy.

ToxicityPiperaquine is safer than chloroquine. It is generally very well tolerated. Dosing is limited by abdominal discomfort. Apart from rare urticarial reactions to DHA occasional abdominal discomfort and diarrhoea have been reported in clinical trials which may have resulted from piperaquine. Although piper-aquine does slightly prolong the electrocardiogram QT interval (approximately similar in magnitude to chloroquine) it is safer than chloroquine in experimental animals and there is no clini-cal evidence for cardiovascular toxicity. No serious adverse effects have been reported.

UseLarge trials in many countries attest to an excellent efficacy and safety profile. DHA-piperaquine has already established itself as an important antimalarial. It is effective against drug-resistant

586 SECTION 9 Protozoan Infections

There are many reports of synergy or antagonism between antimalarial drugs based on isobolograms drawn from in vitro observations. These are often used to justify a particular choice of antimalarial combination but, for the most part, the results are irrelevant to the clinical use of the drugs. Only when synergy or antagonism is extreme, such as the marked synergy between sulphadoxine and pyrimethamine, is this relevant clinically. There are no cases of marked antagonism between the available antimalarial drugs.

Treating MalariaIn severe malaria, parenteral artesunate should be given. Rectal formulations of artemisinin or its derivatives (particularly arte-sunate) offer the possibility of starting treatment in the home or village before referring to the hospital or health centre (Tables 43.8–43.11). Rectal artesunate should become much more widely available in the next few years. For uncomplicated falci-parum malaria artemisinin-based combination treatments (ACTs) are now recommended as first-line treatment every-where. WHO currently recommends one of five ACTs. The choice of partner drug depends on local patterns of sensitivity and cost. For the treatment of P. vivax, P. malariae, P. ovale malaria chloroquine can still be relied upon in many areas although high-level resistance is now well established in Indo-nesia, Micronesia and the island of New Guinea and there are increasing reports of low-level resistance from many parts of Asia and South-America.

ASSESSMENT OF THE THERAPEUTIC RESPONSE

Generally understood and standardized definitions of antima-larial drug treatment responses are important for epidemiologi-cal purposes and helpful in therapeutic decision making. The definitions of severe malaria and treatment failure and the methods of assessing the therapeutic response have all under-gone changes in recent years. In uncomplicated malaria the immediate therapeutic response is usually assessed by the para-site and fever clearance rates or times. WHO definitions of treatment failure are shown in Table 43.12.

Fever Clearance Times (FCT), Parasite Clearance Rates and Times (PCT)The FCT is the interval from beginning antimalarial treatment until the patient is apyrexial. This is easier said than read! Fever does not come down linearly – it often fluctuates erratically. The method and site of measurements should be standardized and the use of antipyretics documented. One approach is to record when temperature first falls below 37.5°C (FCTa) and then when the temperature falls and remains below 37.5°C for 24 hours (FCTb).

The PCT is the interval between beginning antimalarial treatment and the first negative blood slide. The accuracy of the measurement depends on the frequency with which blood slides are taken and the quality of microscopy. The PCT is directly proportional to the admission parasitaemia. The time taken for parasitaemia to fall to half of the admission value (PCT50) and to fall to 10% of the admission value (PCT90) is also a useful comparative measure. In assessing artemisinin responses measurement of the parasite clearance half-life is rec-ommended – this is derived from the slope of the log-linear

FIRST-LINE DRUGS FOR ENDEMIC AREAS

MALARIA DRUG TREATMENTKnown chloroquine sensitive P.

vivax, P. malariae, P. ovale, P. knowlesi, or P. falciparuma

Chloroquine 10 mg base/kg stat. followed by 5 mg/kg at 12, 24 and 36 h; or 10 mg/kg at 24 h, 5 mg/kg at 48 h

or Amodiaquine 10–12 mg base/kg/day for 3 days

Sensitive P. falciparum malariaa

orP. vivax, P. malariae, P. ovale or

P. knowlesia

Artesunate 4 mg/kg/day for 3 days

+ sulfadoxine 25 mg/kg + Pyrimethamine 1.25 mg/kg (SP) single dose

orArtesunate 4 mg/kg/day for 3

days+ Amodiaquine 10 mg base/kg/

day for 3 days as FCTAll including multi-drug

resistant P. falciparum malariaorP. vivax, P. malariae, P. ovale,

or P. knowlesi

Artemether-lumefantrine 1.5/ 9 mg/kg twice daily for 3 days with food

orArtesunate 4 mg/kg/day +

mefloquine 8 mg/kg/day for 3 days in FCT

orDihydroartemisinin-piperaquine

2.5/20 mg/kg/day for 3 daysFCT: Fixed dose combination treatment. In low transmission

settings a single dose of primaquine 0.25 mg base/kg should be added to all falciparum malaria treatments and given with the first dose of ACT, except to infants and pregnant women.

SECOND-LINE TREATMENTSArtesunate 2 mg/kg daily (initial dose may be 4mg/kg) plus either:

(a) tetracycline 4 mg/kg four times daily or (b) doxycycline 3 mg/kg once daily or (c) clindamycin 10 mg/kg twice daily for 7 days

Quinine 10 mg salt/kg three times daily plus either: (a) tetracycline 4 mg/kg four times daily or (b) doxycycline 3 mg/kg once daily or (c) clindamycin 10 mg/kg twice daily for 7 days

Atovaquone-proguanil 20/8 mg/kg once daily for 3 days with food.

RADICAL TREATMENTAfter screening for G6PD deficiency patients with tropical P. vivax

malaria should also be given primaquine 0.5 mg base/kg daily and patients with temperate (long latency) strains or P. ovale infections should be given 0.25 mg base/kg all for 14 days to prevent relapse. In mild G6PD deficiency 0.75 mg base/kg should be given once weekly for 8 weeks. Primaquine should not be given in severe G6PD deficiency or to young infants or pregnant women.

GENERAL POINTSPregnancy: Artemisinin derivatives are currently not recomended

in the 1st trimester. Halofantrine, primaquine, and tetracycline should not be used at any time in pregnancy, and sulfadoxine should not be used very near to term (if alternatives are available).

Vomiting is less likely if the patient’s temperature is lowered before oral drug administration.

Short courses of artesunate or quinine (<7 days) alone are not recommended.

In renal failure the dose of quinine should be reduced by one-third to one-half after 48 hours, and doxycycline but not tetracycline should be prescribed.

The doses of all drugs are unchanged in children and pregnant women.

None of the tetracyclines or doxycycline should be given to pregnant women or children under 8 years of age

aAll ACTs are highly effective against P. vivax, P. malariae, and P. ovale, with the exception of combinations containing SP, as resistance to SP is widespread in P. vivax.

TABLE 43.11 Treatment of Uncomplicated Malaria

43  Malaria 587

phase of parasite decline (a parasite clearance estimator is freely available at http://www.wwarn.org). In areas with fully sensitive parasites, mean values are less than 4 hours.

In vivo Testing of Antimalarial Drug EfficacyThe World Health Organization now recommends that antima-larial drug treatment policy should aim for cure rates of at least 95% and that there should be consideration of policy change if failure rates exceed 10%. Frequent assessment of antimalarial drug efficacy is therefore needed to monitor antimalarial drug resistance and inform policy. In comparative studies, the groups should reflect the population affected by malaria. Too many trials have been conducted in older children or adults in highly endemic areas. These groups have significant background immunity, little or no symptoms and a high rate of self-cure. Drug efficacy is therefore overestimated. The analysis should be age stratified if there is a wide age range included in the study. It is very important that patients, parents or guardians truly understand that participation in a drug trial is voluntary and give informed consent. Ideally pre-treatment with another anti-malarial drug is an exclusion criterion, but in some areas this is very common, in which case, such patients should be included provided details are taken and preferably a baseline blood level is taken.

Design of TrialsAn adequate sample size is required. For example with a sample size of 60 studied patients and six treatment failures, the 95%

confidence interval around the 90% cure rate is 82.4–97.6%. This study is too small as it leaves too much uncertainty as to the true cure rate in the population. In the past antimalarial drug trials have been powered to detect differences between drugs – usually with 95% confidence and 80% power. This is a ‘superiority’ trial. But conducting such trials is increasingly dif-ficult with cure rates over 90% (as they should be) because of the exponential increase in the sample size required. The higher the standard treatment’s cure rate, the more difficult it is to demonstrate conclusively a small difference in favour of a new treatment. An alternative approach is the non-inferiority trial which aims to show that an experimental treatment is ‘not worse’ than the active control (i.e. current treatment) by more than a specified amount – the equivalence margin (often denoted δ). The null hypothesis being tested is that there is a difference between the two groups (i.e. it is the opposite to that in conventional superiority trials) and it is greater than the δ. The main limitation is that confounders introduced in a poorly conducted trial which affect both groups and are unrelated to differences in the efficacy (or toxicity) of the trial regimens, can obscure significant differences. In a superiority trial this might lead to a failure to disprove the null hypothesis – i.e. failure to show difference – but in a non-inferiority trial the direction is opposite; a false rejection of the null hypothesis and conclusion of non-inferiority. This emphasizes the importance in antima-larial drug trials of avoiding errors in drug allocation and administration, poor adherence, errors in end-point ascertain-ment (for antimalarial efficacy this refers particularly to identi-fication of recrudescence) and loss to follow-up.

Blinding is often used to avoid bias in comparative trials although it is often difficult in antimalarial drug assessments because of differences in treatment regimens and the difficulties in masking the taste of the drugs. Compared with superiority trials, blinding does not protect against bias as well in non-inferiority trials because a biased investigator wishing to show non-inferiority can simply give all patients similar results! Anal-ysis of non-inferiority trials requires a calculation of the differ-ence between the failures rates in the treatment groups and a calculation of the confidence interval around this difference using appropriate methods and ‘effective’ sample sizes.

In antimalarial drug trials, data should be entered on a case record form. Baseline clinical and demographic details should be recorded and, at a minimum, the parasitaemia counted and haemotocrit measured. In well-equipped sites, parasite culture to correlate the in vivo response with in vitro susceptibility can also be performed. A baseline whole blood sample (or blood spot on filter paper) should be stored for parasite genotyping. Molecular typing of Plasmodium falciparum (usually by assess-ment of size polymorphisms in fragments of the genes encoding MSP1, MSP2 and GLURP) has considerably improved the accu-racy of drug trials conducted in endemic areas. The genotype(s) of infections recurring within the follow-up period is compared with those in the initial isolate. If the same genotype is found the infection is considered a recrudescence (i.e. a treatment failure). A different genotype indicates a newly acquired infec-tion. The method is not foolproof; genotypes may be difficult to ascribe in mixed infections (which are usual in high trans-mission settings) and resistant infections might be subpatent on admission and therefore be considered erroneously as a new infection when they subsequently recrudesce. But genotyping is a considerable advance which allows community-based studies to be conducted in endemic areas. For studies of slowly

Treatment Outcome Symptoms and Signs

Early treatment failure Development of danger signs or severe malaria on days 1–3 in the presence of parasitaemia

Parasitaemia on day 2 higher than the day 0 count irrespective of axillary temperature

Parasitaemia on day 3 with axillary temperature of ≥37.5°C

Parasitaemia on day 3 of ≥25% of count on day 0

Late treatment failure Late clinical failure Development of danger signs or

severe malaria after day 3 in the presence of parasitaemia, without previously meeting any of the criteria of early treatment failure

Presence of parasitaemia and axillary temperature of ≥37.5°C (or history of fever) on any day from day 4 to day 28, without previously meeting any of the criteria of early treatment failure

Late parasitological failure

Presence of parasitaemia on any day from day 7 to day 28 and axillary temperature of <37.5°C, without previously meeting any of the criteria of early treatment failure or late clinical failure

Adequate clinical and parasitological response

Absence of parasitaemia on day 28 irrespective of axillary temperature without previously meeting any of the criteria of early treatment failure, late clinical failure or late parasitological failure.

TABLE 43.12

WHO Definitions of Antimalarial Treatment Failure in Uncomplicated Falciparum Malaria

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venous bicarbonate and plasma or serum urea or creatinine can also be followed and used as measures of the therapeutic response.

In Vitro Antimalarial Drug Susceptibility TestingBoth in vivo and in vitro assessments of antimalarial efficacy are needed to guide treatment recommendations and planning of policy. P. falciparum can be cultured relatively easily in vitro, whereas the other malaria parasites are more difficult to grow ex vivo. Short-term culture of P. falciparum over one cycle requires only simple sterile culture media, a candle jar and an incubator. Short-term culture of P. vivax is also relatively easy with modifications to the conditions. Antimalarial drug suscep-tibility can be tested by measuring the inhibition by different concentrations drugs of parasite maturation to the schizont stage, the degree of inhibition of radio-labelled 3H-hypoxan-thine uptake or sybr green nucleic acid staining, or the synthesis of parasite-specific lactate dehydrogenase or histidine-rich protein 2. The PfLDH and PfHRP2 tests require only an ELISA reader and have the additional advantage of being possible at low parasite densities. These are useful epidemiological tools, but they do not predict the clinical response to treatment in an individual because they do not reflect differences between people in antimalarial pharmacokinetics, immunity or stage of disease.

Patient ManagementIn many tropical countries ‘malaria’ is synonymous with ‘fever’. Antimalarial drugs are self-administered on a vast scale. Where possible, a definite species diagnosis should be obtained by microscopy examination of the blood smear or use of a suitable rapid antigen-based diagnostic test (RDT). If there is any doubt, Plasmodium falciparum infection should be assumed. The man-agement of malaria depends very much on the health facilities available and the endemicity of disease, i.e. the likely immune status of the patient. For example, in areas of intense transmis-sion infants and young children are often parasitaemic. Distin-guishing malaria from other infections as the cause of fever may be difficult or impossible and so all febrile parasitaemic children must be treated for malaria unless there is an evident alternative diagnosis. In these settings asymptomatic parasitaemia is also common in older children and adults, but in these age groups fever is more likely to be the result of some other infection. On the other hand fever may precede detectable parasitaemia in non-immune adults or young children. The blood film should be rechecked in suspected cases. ‘Blood smear-negative malaria’ is a common diagnosis in the tropics – but one to be avoided. Other infections are more likely. If the patient has a sub-patent parasitaemia and no signs of severity it is safe to wait, seek other causes for the symptoms and repeat the blood smears at 12–24-hour intervals. In severely ill patients antimalarial treatment should be started immediately in full doses, but other diagnoses sought. Patients may remain unconscious or develop renal failure after parasite clearance, but there is usually a clear history of previous treatment and malaria pigment may still be found in monocytes in peripheral blood or intradermal smears and the Pf HRP2 dipstick will be positive. If the temperature is high on admission (>38.5°C) then symptomatic treatment with oral antipyretics (paracetamol, not aspirin) and tepid sponging brings symptomatic relief and may also reduce the likelihood that the patient vomits the oral antimalarials. This is

eliminated drugs, taking a blood level measurement at day 7 in all patients helps to interpret treatment failures (i.e. whether they resulted from drug resistance or low blood concentra-tions). For many drugs, simple filter paper whole blood assays are now available.

Antimalarial treatment should be observed and adverse effects recorded. The patients should be followed daily until parasite clearance, then at weekly intervals. The rate of resolu-tion of anaemia is a sensitive measure of the treatment response. The haemoglobin or haematocrit should be measured each time a parasite count is performed in therapeutic assessments. Four weeks is the minimum follow-up duration for rapidly eliminated drugs and 6 weeks is the minimum for drugs with intermediate or long terminal elimination half-lives. At least 90% follow-up at 4 weeks should be aimed for and sample sizes adjusted for likely ‘drop-out’ rates. The appearance of P. vivax, P. malariae or P. ovale malaria requires chloroquine treatment. Whether such patients should be excluded from the analysis in a falciparum malarial trial depends on the level of chloroquine resistance in P. falciparum and needs to be decided before the trial.

Interpretation of TrialsIn antimalarial drug trials two or more groups of patients are followed for a prespecified time after different antimalarial treatments. The cure rates, which means the proportions of patients who reach the end of this follow-up period without recrudescence of the infection, are compared. In the past, anti-malarial treatment efficacy was usually assessed on a particular day (often day 14 or day 28 after starting treatment), so only patients followed to that day were included in the analysis. This is often referred as a ‘per-protocol’ (PP) analysis. But in most trials, there are patients who do not complete the follow-up period, yet these patients do contribute useful information before they leave the trial and this can and should be used. If such a patient did not fail (i.e. remained aparasitaemic) when last observed, that patient’s data are said to be ‘censored’ at the time they were last followed up. The appropriate analysis for such data is survival analysis which deals explicitly with cen-sored values. Patients with different follow-up periods cannot be treated the same way – someone who is followed-up for longer has a greater chance of being recorded as treatment failure than another patient followed up for a shorter time. Failure rates should be estimated using the Kaplan–Meier method. This is now endorsed by the recent WHO recommen-dations for antimalarial resistance monitoring which suggest use of life tables (i.e. survival analysis) in analysing in vivo studies. The ‘intention to treat’ (ITT) analysis which includes all missing patients or indeterminate values as treatment failures should be reported also, but it should not be the primary end-point of a study as it overestimates the true failure rates.

Severe Malaria TrialsIn addition to parasite and fever clearance the rate of clinical recovery in survivors should be assessed. In unconscious chil-dren the Blantyre Coma Scale (BCS) is most widely used and in adults the Glasgow Coma Score (GCS) should be measured. If possible these should be assessed 4–6-hourly and the times to reach BCS scores of 3, 4 and 5 and GCS scores of 8, 11 and 15 recorded. The time to drink, sit, walk and leave hospital should also be documented. The changes in venous lactate,

43  Malaria 589

hospitalized. The choice of drugs will depend on the local pattern of resistance where the infection was acquired. Because of the propensity for P. falciparum infections to kill, careful assessment of severity is most important. There is obviously a distribution of severity from asymptomatic parasitaemia to ful-minant lethal malaria. In practice any patient who is unable to take oral medication will require parenteral treatment and careful observation and any impairment of consciousness should be treated seriously. The progression to cerebral malaria can be rapid, particularly in young children.

MANAGEMENT OF SEVERE P. FALCIPARUM MALARIA

Severe malaria is a medical emergency.27 (See Table 43.13 for immediate clinical management.) The airway should be secured in unconscious patients, an intravenous infusion should be started and other resuscitation measures taken. A rapid clinical assessment of the degree of dehydration and the intravascular volume should be made. Vital signs and capillary refill time should be recorded. Particular attention should be paid to the respiratory pattern and any signs of respiratory distress (laboured deep breathing, flaring of the alar nasae, intercostal or substernal retraction) should be noted. The patient should be weighed or weight assessed so that the antimalarials can be given on a body weight basis (for adults, a simple method is for the stretcher-bearers to stand on bathroom scales with and without, the patient). Immediate measurements of blood glucose (stick test), haematocrit, parasitaemia (parasite count, stage of development and proportion of neutrophils containing malaria pigment) and renal function (blood urea or creatinine) should be taken. The degree of acidosis is an important deter-minant of outcome; the plasma bicarbonate or venous lactate should be measured if possible (lactate rapid stick tests are now available). Arterial or capillary blood pH and gases should be measured in patients who are unconscious, hyperventilating, or shocked. Blood should be taken for cross-match and later (if available) full blood count, platelet count, clotting studies, blood culture and full biochemistry. Parenteral antimalarial treatment should be given as soon as possible. Where there are adequate nursing facilities the antimalarial drugs should be given by intravenous infusion. There is no specific treatment for severe malaria other than antimalarial drugs. These are poten-tially life-saving and so it is very important that the dosing is correct (the first dose is by far the most important). Artesunate should be given by intravenous or intramuscular injection and artemether by intramuscular injection only. If quinine is used a full loading dose (20 mg dihydrochloride salt/kg) should be given to all patients unless there is a clear history of adequate pretreatment. Quinine is compatible with saline or dextrose solutions. It should never be given by bolus intravenous injec-tion. In African children with severe malaria sepsis usually cannot be excluded reliably and so empirical broad-spectrum antibiotics should be given from the outset until a bacterial infection can be excluded.

The assessment of fluid balance is critical in severe malaria. In children there is not a consensus as to optimum fluid man-agement. Some children are clearly ‘dry ‘on admission and need rehydration but rapid fluid loading has proved clearly harmful. Urgent blood transfusion is required for severely anaemic (hae-matocrit <15%) acidotic children, but the role of colloids oth-erwise remains controversial. In adults there is a thin dividing

particularly important for young children who are less likely to have a seizure and more likely to tolerate oral antimalarials when their temperature has been lowered and they are quiet and calm. Unfortunately, there are no paediatric formulations for some of the ACTs. For young children large pills should be crushed and given as a suspension in a small volume of sweet drink or milk when treating young children. A disposable syringe (without the needle!) may be used to draw up and give an accurate volume of the suspension into the child’s mouth.

P. VIVAX, P. OVALE OR P. MALARIAE

Although P. vivax, P. ovale or P. malariae rarely kill, the disease can be moderately severe, requiring initial parenteral treatment. Occasional patients with vivax malaria do develop vital organ dysfunction and these should be treated as for severe falci-parum malaria. More usually oral treatment with chloroquine (Table 43.11) leads to resolution of the fever within 2–3 days. The total dose is usually 25 mg base/kg. The initial dose is 10 mg base/kg and this is followed at 12-hour intervals with subsequent doses of 5 mg/kg or the dose is divided as 10, 10, 5 mg/kg on days 0, 1 and 2, respectively. ACTs are a highly effec-tive alternative (with the exception of artesunate – SP in some areas). Resistance to SP is widespread and high-level resistance to chloroquine in P. vivax is now a significant problem on the island of New Guinea and in parts of Indonesia. These infec-tions respond to piperaquine or mefloquine containing ACTs. Plasmodium vivax responds to antimalarial drugs similarly to Plasmodium falciparum. After screening for G6PD deficiency a full radical course of primaquine should be given to all patients with P. vivax or P. ovale to prevent relapse. The incidence of relapse varies considerably by geographic region and transmis-sion intensity. The efficacy in preventing relapse is determined by the total dose of primaquine taken. The 5-day regimens widely used on the Indian subcontinent were insufficient. The recommended dose is 0.5 mg base/kg per day for 14 days although the lower dose of 0.25 mg base/kg per day is reliably effective against long latency temperate strains. Primaquine is often considered unnecessary if the patient is going to return immediately to a highly endemic area, although the risk–benefit assessment for use in children in Asia (where G6PD deficiency is common) has not been made. Primaquine should not be given to pregnant or lactating women or infants or patients with known severe variants of G6PD deficiency. If mild variants of G6PD deficiency are known or likely then primaquine can be given in a dose of 0.75 mg/kg (45 mg) once weekly for 8 weeks. Primaquine does have significant activity against the asexual blood stages of P. vivax and this may mask chloroquine resis-tance in combined treatment, but may also protect against chlo-roquine resistance in areas with sensitive parasites.

P. KNOWLESI MALARIA

This should be treated in the same way as acute falciparum malaria with ACTs or artesunate for severe malaria. P. knowlesi is sensitive to chloroquine.

P. FALCIPARUM MALARIA

In endemic areas uncomplicated falciparum malaria is treated on an outpatient basis in the same way as the other malarias. In temperate countries imported cases should usually be

590 SECTION 9 Protozoan Infections

directly and maintained between 0 and 5 cm. However, recent studies shows that the CVP correlates poorly with other hae-modynamic indices and is thus a poor guide to fluid replace-ment. If the venous pressure is elevated (usually because of over-enthusiastic fluid administration) and the patient becomes breathless they should be nursed with the head at 45° and if necessary intravenous frusemide given. Acidotic breathing or respiratory distress, particularly in severely anaemic children may indicate hypovolaemia and requires prompt rehydration and, if available, urgent blood transfusion. Convulsions should be treated promptly with intravenous or rectal lorazepam (or if unavailable diazepam or midazolam) or intramuscular paralde-hyde. The role of prophylactic anticonvulsants is unresolved (see below).

When these immediate measures have been completed a more detailed clinical examination should be conducted, with particular note of the level of consciousness and record of the coma score. Several coma scores have been advocated. The Glasgow Coma Scale (GCS) is suitable for adults and the simple Blantyre modification (BCS) is readily performed in children (Table 43.14). Unconscious patients must have a diagnostic lumbar puncture to exclude bacterial meningitis. The opening pressure should be recorded and the rise and fall with respira-tion noted. The CSF should be sent for microscopy examina-tion, culture and measurement of glucose, lactate and protein. Subsequent clinical observations should be as frequent as pos-sible and should include vital signs, with an accurate assessment of respiratory rate and pattern, assessment of the coma score and urine output. The blood glucose should be checked, using rapid stick tests every four hours if possible, until recovery of consciousness. These stick tests may overestimate the frequency of hypoglycaemia so laboratory confirmation may be necessary. Important milestones on the road to recovery are the time to recover consciousness (GCS 15 or BCS 5), time to drink and times to sit unaided and walk.

CEREBRAL MALARIA

When managing a patient who is unconscious with severe malaria, the physician must exclude, as far as possible, continu-ous seizure activity and hypoglycaemia as the cause. Both are more common in children than in adults. Many adjuvant therapies have been suggested, based on the prevailing patho-physiology hypotheses of the time. These include heparin, low-molecular-weight dextran, urea, high-dose corticosteroids, aspirin, prostacyclin, pentoxifylline (oxpentifylline), desferriox-amine, anti-TNF antibody, cyclosporine, hyperimmune serum, mannitol, albumin and saline fluid loading. Unfortunately none has proved to be beneficial and many have proved harmful. None of these adjuvants should be used. The cornerstone of management is good intensive care and prompt appropriate antimalarial treatment.

Prophylactic phenobarbitone prevents seizures in cerebral malaria. But the role of prophylactic anticonvulsants is uncer-tain since a large double-blind trial in Kenyan children with cerebral malaria (and no access to mechanical ventilation) showed a doubling of mortality in children receiving a single prophylactic intramuscular injection of phenobarbitone (20 mg/kg). Mortality was increased in children who received three or more doses of diazepam (i.e. had recurrent treated seizures), which suggests a possible interaction between these two drugs and points to respiratory depression as the lethal

Manifestation/Complication Immediate Managementa

Coma (cerebral malaria) Maintain airway, place patient on his or her side, exclude other treatable causes of coma (e.g. hypoglycaemia, bacterial meningitis); avoid harmful ancillary treatment such as corticosteroids, fluid loading, mannitol, heparin and adrenaline; intubate if necessary.

Hyperpyrexia Administer tepid sponging, fanning, cooling blanket and antipyretic drugs.

Convulsions Maintain airways; treat promptly with intravenous or rectal lorazepam, diazepam or intramuscular paraldehyde.

Hypoglycaemia (blood glucose concentration of <2.2 mmol/L)

Check blood glucose, correct hypoglycaemia and maintain with glucose-containing infusion.

Severe anaemia (haemoglobin <5 g/100 mL or packed cell volume <15%)

Transfuse with screened fresh whole blood.

Acute pulmonary oedemab Prop patient up at an angle of 45°, give oxygen, give a diuretic, stop intravenous fluids, intubate and add positive end-expiratory pressure/continuous positive airway pressure if hypoxaemic

Acute kidney injury Exclude pre-renal causes, check fluid balance and urinary sodium; if in established renal failure start haemofiltration or haemodialysis, or if unavailable, peritoneal dialysis. The benefits of diuretics/dopamine in acute renal failure are not proven.

Spontaneous bleeding and coagulopathy

Transfuse with screened fresh whole blood (cryoprecipitate, fresh frozen plasma and platelets if available); give vitamin K injection.

Metabolic acidosis Exclude or treat hypoglycaemia, hypovolaemia and septicaemia. If severe start haemofiltration or haemodialysis.

Shock Suspect septicaemia, take blood for cultures; give parenteral antimicrobials, correct haemodynamic disturbances.

aIt is assumed that appropriate antimalarial treatment will have been started in all cases.

bPrevent by avoiding excess fluid administration.

TABLE 43.13

Immediate Clinical Management of Severe Manifestations and Complications of Falciparum Malaria

line between overhydration which may precipitate pulmonary oedema and underhydration which may contribute to shock or precipitate or worsen acidosis and renal impairment. Careful and frequent evaluations of the jugular venous pressure, periph-eral perfusion, venous filling, skin turgor and urine output should be made. Where there is uncertainty over the jugular venous pressure and if nursing facilities permit, a central venous catheter may be inserted and the pressure (CVP) measured

43  Malaria 591

unconscious for as long as 10 days. With longer periods of coma complications such as pressure sores and secondary infections become increasingly likely.

FLUID BALANCE

Children with severe malaria may be dehydrated, but renal failure and pulmonary oedema are extremely unusual in young children. A common mistake is to be too cautious in giving blood to an anaemic acidotic child for fear of precipitating ‘congestive failure’. Anaemic congestive failure is uncommon and that respiratory distress in these children represents meta-bolic acidosis not pulmonary oedema. The acidosis is aggra-vated by severe anaemia and sometimes hypovolaemia. However the concept that hypovolaemia is an important cause of death has been challenged by the clear demonstration that fluid loading with either crystalloid or colloid increases mortality. This argues for a more cautious approach to fluid resuscitation in children as well as adults. In approximately 50% of adults admitted with severe malaria there is evidence of renal impair-ment. In the majority of these there will be a transient period of oliguria, followed by uncomplicated recovery, but a minority will progress to established acute tubular necrosis. A polyuric phase is unusual. Adults with severe malaria are very vulnerable to fluid overload and the physician treads a narrow path between underhydration and thus worsening renal impairment and overhydration, with the risk of precipitating pulmonary oedema. Following admission patients should be rehydrated carefully with 0.9% (normal) saline or other isotonic electrolyte solu-tions. Thereafter the daily fluid requirements will depend on urine output (plus diarrhoea) and insensible losses, which can be considerable in febrile patients nursed in hot environments. Water and glucose are provided by 5% or 10% dextrose solutions. Hypoglycaemic patients will often require 10% dex-trose infusions after a bolus glucose correction. It is not possible

effect. Thus the standard loading dose of phenobarbitone is contraindicated unless the patient can be ventilated. Some phy-sicians give a smaller dose of phenobarbitone in unconscious patients, others do not give any seizure prophylaxis and rely on treatment. The safety and effectiveness of phenytoin, fosphe-nytoin and other anticonvulsants is not well characterized. Although approximately 80% of children with cerebral malaria have moderately elevated pressures at lumbar puncture (whereas in adults 80% of pressures are in the normal range) and some children have very high pressures, use of the osmotic agent mannitol proved harmful in a recent trial conducted in adults. Those factors known to exacerbate raised intracranial pressure such as uncontrolled seizures and hypercapnoea should be treated promptly. Specific management includes care of the unconscious patient, careful fluid balance, rapid treatment of convulsions, treatment of hyperpyrexia and early detection and treatment of other manifestations or complications of severe malaria.

Hypoglycaemia should be suspected in any patient who deteriorates suddenly and this should be treated empirically if glucose stick tests are unavailable. Supervening bacterial infec-tions are common, particularly chest infections and catheter-related urinary tract infections and spontaneous septicaemia may occur occasionally. Bacteraemia is much more common in African children than in adults or children studied in South-East Asia. There is undoubtedly diagnostic overlap between severe malaria and bacterial septicaemia with incidental para-sitaemia, but there is also a genuine predisposition to septicae-mia in severe malaria. If not already given empirical broad-spectrum antibiotics should be given to any patient who deteriorates suddenly and in whom hypoglycaemia has been excluded. Aspiration pneumonia commonly follows generalized seizures. Patients should be nursed on their sides and turned frequently. Most children will recover consciousness within 2 days and most adults within 3 days. Rarely adults may remain

The Blantyre Coma Scale for Children The Modified Glasgow Coma Scale for Adults

Scorea Scoreb

BEST MOTOR RESPONSE BEST MOTOR RESPONSELocalizes painful stimulusc 2 Obeys commands 5Withdraws limb from paind 1 Localizes pain 4Nonspecific or absent response 0 Flexion to pain 3

Extension to pain 2None 1

VERBAL RESPONSEe 2 VERBAL RESPONSEAppropriate cry 1 Oriented 5Moan or inappropriate cry 0 Confused 4None Inappropriate words 3

Incomprehensible sounds 2None 1

EYE MOVEMENTS EYE OPENDirected (e.g. follows mother’s face) 1 Spontaneously 4Not directed 0 To speech 3

To pain 2Never 1

aTotal score can range from 0 to 5; 2 or less indicates ‘unrousable coma’.bTotal score can range from 3 to 14; ‘Cerebral malaria defined by GCS <11; Unrousable coma’ reflects a score of <9.cPainful stimulus: rub knuckles on patient’s sternum.dPainful stimulus: firm pressure on thumbnail bed with horizontal pencil.eNot readily assessed in preverbal children.

TABLE 43.14 Coma Scales to Assess Levels of Consciousness in Adults and Children

592 SECTION 9 Protozoan Infections

used to remove excess fluid and also to provide glucose in hypoglycaemic cases. The efficiency of peritoneal dialysis often improves after the first 24 hours. Reduced peritoneal clearance is thought to be related to sequestration in the peritoneal micro-vasculature during the acute phase. Peritonitis (cloudy dialysis effluent) is relatively common if dialysis is continued for more than 72 hours. The dose of quinine should be reduced by between one-third and one-half on the 3rd day of treatment. Tetracycline is contraindicated, but doxycycline can still be given. The median time to recovery of urine flows (>20 mL/kg per 24 hours) is 4 days. The overall prognosis and rate of recov-ery is better in oliguric than in anuric cases (Figure 43.29).

Patients with blackwater fever should be managed in the same way as other patients. Parenteral antimalarial treatment should continue. The preventative or therapeutic role of urinary alkalinization has not been evaluated yet. Blood transfusion is often needed but the increase in haematocrit is often less than predicted because of the brisk haemolysis of the transfused cells. If the patient is volume overloaded, but needs blood, then dialysis or haemofiltration must be given first to create enough vascular ‘space’ for the blood. Packed cells should be given and the transfusion administered as slowly as possible.

ACUTE PULMONARY OEDEMA

This grave manifestation of severe malaria commonly co-exists with acute renal failure. The differential diagnosis includes pneumonia, if there are abnormal chest signs and metabolic acidosis, if the chest is clear. Frothing at the mouth does not necessarily mean acute pulmonary oedema. In children, it is often hypersalivation resulting from continuous seizures. Tachypnoea is a serious sign in malaria; occasionally it results from high fever alone, in which case breathing is shallow, but more usually there is noisy hyperventilation with use of acces-sory muscles of respiration, intercostal recession and flaring of the nostrils. Patients with acute pulmonary oedema should be nursed upright and given oxygen and the right-sided filling pressures should be reduced with whichever treatments are available (loop diuretics, opiates, venodilators, venesection, haemofiltration, dialysis). The right-sided pressure should be

to generalize on initial fluid requirements as these can vary from deficits of several litres, to patients who are admitted oliguric and unconscious on a saline infusion and are well hydrated with a slightly elevated jugular venous pressure. Each patient’s requirements should be assessed individually. If there is no CVP line it is well worth spending some time establishing clearly the level of the jugular venous pressure. If blood glucose is <4 mmol/L then 10% glucose should be started following saline replacement; if it is <2.2 mmol/L then hypoglycaemia should be treated immediately (0.3–0.5 g/kg of glucose). The fluid regimen must also be tailored around infusion of the antima-larial drugs. Artesunate and artemether are simple injections but quinine infusions must be rate-controlled. Some physicians prefer to put the 24-hour quinine maintenance dose in one 500 mL bottle of 0.9% saline or 5% dextrose water and infuse this at constant rate, while adjusting fluid balance as necessary through a separate piggy-backed line. It is rarely necessary to give potassium or other electrolyte supplements in the acute phase. Many patients will require blood transfusion. The exact criteria for transfusion will depend on blood availability, but in general if the haematocrit falls below 20% then blood should be given, although in high transmission settings this would necessitate too many transfusions. The lower threshold of 15% haematocrit is often used. In adults with severe malaria where there is a greater danger of precipitating pulmonary oedema, transfusion of packed cells may be indicated. In practice, if blood is allowed to sediment in a bag or bottle, only the cells can be given. If the patient is volume overloaded the transfusion should be stopped, or continued very slowly, adding frusemide (0.3 mg/kg) to each unit.

ACUTE KIDNEY INJURY

If the patient remains oliguric (<0.4 mL of urine/kg per hour) despite adequate rehydration and the blood urea or creatinine are rising or already high, then fluids should be restricted to replace insensible losses only. Dialysis or haemofiltration renal replacement therapies (RRT) should be started early when there is evidence of multiple organ dysfunction. There is no evidence that use of dopamine and loop diuretics prevents the progres-sion of renal failure. Renal impairment is hypercatabolic in the acute phase of the disease and once conventional indications for RRT have been reached (i.e. metabolic acidosis, uraemic complications, volume overload, or less commonly hyperkalae-mia) the patient may deteriorate quickly. An electrocardiogram should be performed if acute renal failure is suspected and an immediate blood potassium measurement is unavailable. If there are signs of hyperkalaemia (peaked T waves, widening of the QRS complex) then calcium and glucose plus insulin, should be given immediately. The tempo of disease is faster in patients with acute disease and multiple organ dysfunction and RRT should be started earlier than in those whose renal failure develops after other acute manifestations have resolved. Hae-mofiltration or haemodialysis are preferable to peritoneal dialy-sis. Haemofiltration is associated with a considerably more rapid resolution of biochemical abnormalities and a lower mor-tality than peritoneal dialysis. Despite the coagulopathy associ-ated with severe malaria bleeding problems are unusual. After the initial outlay for the pumps and balance haemofiltration is also less expensive, although well-trained nursing care is essen-tial. When there is no alternative to peritoneal dialysis the addi-tion of hypertonic dextrose to the peritoneal dialysate can be

Figure 43.29 Recovery from malaria acute renal failure. This results from acute tubular necrosis. Many patients will not become oliguric, despite a rising serum creatinine in the first few days of hospitalization and can be managed conservatively. (From Trang et al. Clin Infect Dis 1992;15:874–880.)

Days

Peritoneal dialysis

0 5 10 15 20 25 30

Seru

m c

reat

inin

e (µ

mol

/L)

0

1000

800

600

400

200

43  Malaria 593

pneumonia should be given empirical treatment with a third-generation cephalosporin, unless admitted with clear evidence of aspiration, in which case penicillin or clindamycin is ade-quate. Children with persistent fever despite parasite clearance may also have a systemic Salmonella infection, although in the majority of cases of persistent fever after parasite clearance no other pathogen is identified. Urinary tract infections are common in catheterized patients. Antibiotic treatments should depend on likely local antibiotic sensitivity patterns. Sustained high fever in the acute phase of severe malaria is a poor prog-nostic sign. Continued fever after parasite clearance is common; antibiotic treatment is only indicated if the patient becomes severely ill or there is a definite focus of infection.

TREATMENT OF RECRUDESCENT INFECTIONS

Treatment failure within 14 days of receiving an ACT is very unusual. Thus the majority of treatment failures occur more than 2 weeks after treatment. In many cases failures are not recognized because patients presenting with malaria are not asked whether they have received antimalarial treatment within the preceding 1–2 months. Recurrence of malaria can be the result of a reinfection, a recrudescence (i.e. failure) or, a relapse malaria due to P. vivax and P. ovale, although relapses do not appear within 14 days of the primary infection. In an individual patient it is initially not possible to distinguish recrudescence from reinfection. Wherever possible, treatment failure must be confirmed parasitologically – preferably by blood slide exami-nation, as the HRP2-based stick tests may remain positive for weeks after the initial infection even without recrudescence. Treatment failures may result from drug resistance, poor adher-ence or unusual pharmacokinetic properties in that individual. It is important to determine from the patient’s history whether he or she vomited previous treatment or did not complete a full course. Recurrence of fever and parasitaemia more than 2 weeks after treatment (either recrudescence or new infection), can be retreated with the first-line ACT. If it is a recrudescence, then the first-line treatment should still be effective in most cases. This simplifies operational management and drug deployment. However, reuse of mefloquine within 28 days of first treatment is associated with an increased risk of neuropsychiatric sequelae and, in this particular case, second-line treatment should be given. If there is a further recurrence, then malaria should be confirmed parasitologically and second-line treatment given. The following second-line treatments are recommended by WHO, in order of preference:34

• Alternative ACT known to be effective in the region• Artesunate plus tetracycline or doxycycline or clindamycin

(7 days)• Quinine plus tetracycline or doxycycline or clindamycin

(7 days).The alternative ACT has the advantages of simplicity, familiarity and, where available, coformulation to improve adherence. The 7-day quinine regimens are not well tolerated and adherence is likely to be poor if treatment is not observed.

Malaria in PregnancySEVERE MALARIA

Pregnant women in the 2nd and 3rd trimesters are more likely to develop severe malaria than other adults, often complicated

reduced to the lowest level compatible with an adequate cardiac output. Positive pressure ventilation should be started if the patient becomes hypoxic.

HYPOGLYCAEMIA

There should be a low threshold for suspecting hypoglycaemia. There may be no signs of hypoglycaemia in a patient already unconscious with cerebral malaria. Ideally, blood glucose should be checked 4-hourly while patients are unconscious. Hypogly-caemia should be treated by slow intravenous injection of 0.5–1 mL/kg of 50% dextrose water and prevented by adminis-tering a 10% dextrose infusion at 1–2 mg/kg per hour. Quinine-stimulated hyperinsulinaemia may be blocked by somatostatin or its synthetic analogue, if available (they seldom are!). If pos-sible, serum potassium should be checked frequently in hypo-glycaemic patients receiving quinine and hypertonic dextrose solutions.

ACIDOSIS

Hypovolaemia should be corrected although recent evidence suggest that this is not a major contributor to acidosis. The circulatory status is more difficult to assess in children than in adults. But in many cases the acidosis persists despite adequate blood pressure, adequate capillary refill and warm peripheries and normal jugular venous pressure. Although acidosis may result from acute renal failure, ketonaemia and even salicylate poisoning, in most cases lactic acidosis is a significant contribu-tor to the wide anion gap. Venous, arterial and CSF concentra-tions of lactate rise in proportion to disease severity and in contrast to sepsis, they are associated with an increased lactate–pyruvate ratio (often >30) indicating anaerobic glycolysis. Lactic acid accumulation is buffered initially, but decompensa-tion often occurs in severe malaria. The role of sodium bicar-bonate in the treatment of metabolic acidosis has declined from established practice to the controversial. Now most authorities either do not give sodium bicarbonate at all, or give it once only in very severe acidosis (e.g. pH <7.15). The pyruvate dehydro-genase activator dichloroacetate has proved promising in pre-liminary clinical trials, but its role in treatment remains to be defined. Haemofiltration or haemodialysis may be used to control acidosis.

BLEEDING

Patients with cerebral malaria may have haematemesis or a bloody nasogastric aspirate because of acute gastric erosions. The incidence of upper gastrointestinal bleeding has declined since the discontinuation of high-dose corticosteroids in cere-bral malaria. The role of prophylactic antacids, H2 blockers, sulfacrate or proton pump inhibitors has not been studied spe-cifically in severe malaria. Less than 5% of patients with severe malaria develop clinically significant DIC. These patients should be given fresh blood transfusions and vitamin K.

BACTERIAL SUPERINFECTION/CONTINUED FEVER

The treatment of suspected septicaemia will depend on local antimicrobial susceptibility patterns, bearing in mind that Salmonellae may be implicated. Patients with secondary

594 SECTION 9 Protozoan Infections

concerns over mefloquine in treatment use, although there is no significant evidence of an increased stillbirth risk when used as prophylaxis. Primaquine and doxycycline are contraindi-cated. Preliminary evidence suggests that atovaquone-proguanil is safe.

INTERMITTENT PREVENTIVE TREATMENT (IPTp)

Studies conducted in high transmission areas of Africa have shown that administration of treatment doses of sulfadoxine-pyrimethamine (SP) two or three times during pregnancy was associated with reduced placental parasitization, reduced anaemia and increased birth weight. IPTp with SP has been increasingly adopted but since the original studies were con-ducted resistance has worsened considerably in Africa compro-mising efficacy. A review of available information suggests continuing efficacy in many areas but has led to revised recom-mendations that IPTp SP should be given more frequently (maximum monthly). Alternative drugs are under evaluation.

BREAST-FEEDING

Nearly all the antimalarial drugs appear in breast milk, but the actual amounts excreted are small. Primaquine should be avoided, pending further information, but otherwise there seems no reason to discourage breast-feeding in women receiv-ing antimalarial drugs.

Malaria in ChildrenAlthough maternal malaria is very common, congenital malaria is surprisingly rare given the high frequency with which placen-tal smears are positive in endemic areas and the not infrequent finding of parasites in cord blood smears. Nevertheless, it may occur with any of the four human malarias. Congenital falci-parum malaria is seldom severe. Congenital P. vivax or P. ovale infections do not require radical treatment as there are no pre-erythrocytic stages in the baby.

Severe malaria is relatively uncommon in the first 6 months of life, although when it does occur the mortality is high. In young children malaria presents as a febrile illness without focal signs. In P. falciparum infections, convulsions are an important complication in the first 3 years. They are twice as common as in P. vivax malaria, despite similar fever profiles. The progres-sion to cerebral malaria in young children can be very rapid. Recovery is also rapid compared with adults. In areas of intense transmission, severe anaemia in the 1–3-year age group is the principal manifestation of severe falciparum malaria and may also occur with repeated vivax infections. A comparison of the relative frequencies of complications in adults and children is shown in Table 43.15.

Children receive the brunt of malaria’s assault on humans. Most of the deaths from malaria are in children and most of those are in Africa. Malaria is also an important cause of mor-bidity, failure to thrive and probably increased susceptibility to other infections. Whether cerebral malaria, malaria-associated convulsions, or the debilitating effects of repeated weakening febrile illnesses and anaemia cause developmental or intellec-tual retardation needs to be determined. There is evidence for learning difficulties in children who have had seizures in malaria and in cerebral malaria survivors. In general children tolerate the antimalarial drugs better than adults. In severe malaria fluid

by pulmonary oedema and hypoglycaemia. Fetal death and pre-mature labour are common. The role of early caesarean section for the viable live fetus is unproven, but recommended by many authorities. Obstetric advice should be sought at an early stage, the paediatricians alerted and the blood glucose checked fre-quently. Hypoglycaemia should be expected and is often recur-rent if the patient is receiving quinine. Artesunate is safer and more effective. The antimalarial drugs should be given in full doses. Severe malaria may also present immediately following delivery. Postpartum bacterial infection is a common complica-tion in these cases. Falciparum malaria has also been associated with severe mid-trimester haemolytic anaemia in Nigeria. This often requires transfusion, in addition to antimalarial treatment and folate supplementation.

UNCOMPLICATED MALARIA

Symptomatic malaria in pregnancy requires hospitalization where possible. Premature labour may occur and pregnant women receiving quinine are liable to develop hypoglycaemia. Chloroquine, pyrimethamine, proguanil, mefloquine, quinine and the sulphonamides are considered safe in pregnancy. Amodiaquine has been widely used but not well documented. The artemisinin derivatives are safe in the 2nd and 3rd trimes-ters, but there is still some uncertainty in the 1st trimester, where they are currently not recommended. There is increas-ing confidence in the safety of artemether-lumefantrine and some preliminary experience with atovaquone-proguanil and dihydroartemisinin-piperaquine indicating that these drugs are also safe. The tetracyclines and primaquine are contraindicated. As a consequence the five first-line ACTs are recommended for the treatment of falciparum malaria in the 2nd and 3rd trimes-ters. Quinine (10 mg salt/kg three times daily for 7 days) is still the treatment of choice for falciparum malaria in the 1st trimes-ter but it is poorly tolerated and low adherence leads to high treatment failure rates. The artemisinin derivatives and quinine should both be combined with clindamycin (10 mg/kg twice daily) to increase cure rates. Treatment failure rates are higher in pregnant women than in non-pregnant adults for any anti-malarial regimen. Pharmacokinetic studies indicate that blood concentrations of the artemisinin derivatives, lumefantrine, atovaquone and proguanil are all significantly reduced in late pregnancy, so current dose recommendations may not be optimal. Data on sulfadoxine-pyrimethamine are variable and both mefloquine and piperaquine pharmacokinetics are not significantly altered. Close follow-up of pregnant women is essential. Women in malarious areas should be encouraged to attend weekly antenatal clinics where blood smears and hae-matocrit can be checked, in addition to routine obstetric assessment.

PreventionPROPHYLAXIS

If effective in the area and safe, antimalarial prophylaxis should be given during pregnancy. Chloroquine (5 mg base/kg per week) is generally still very effective in preventing P. vivax. P. ovale and P. malariae. Unfortunately, P. falciparum is usually present at the same time and nearly always resistant. In areas where P. falciparum is still sensitive to antifols daily proguanil (3.5 mg/kg per day) is safe and effective. There have been some

43  Malaria 595

PreventionINSECTICIDE-TREATED BED NETS

The chances of being bitten by a malaria-infected female anoph-eline mosquito can be reduced considerably by simple measures. Covering exposed skin surfaces and remaining indoors or under a net at peak biting times will obviously reduce exposure. For example, most mosquitoes feed at night so sleeping indoors under insecticide (permethrin, deltamethrin)-treated bed nets reduces morbidity and mortality in malarious areas. A single impregnation of a cotton or nylon mosquito net will provide protection for 1 year. Nylon tends to retain permethrin and deltamethrin better than cotton. The impregnated bed nets (ITN) can be washed and can tolerate small tears or holes without markedly reducing the protective effects. Now ‘long-lasting impregnated nets’ (LLIN) have been developed which retain insecticidal activity for many years. These are more expensive but may be cost-effective. At the time of writing four types of LLINs have full endorsement and nine have interim endorsement by the WHO Pesticide Evaluation Scheme. The benefits conferred by bed-nets depend greatly on the biting habits of the mosquito, the size and constitution of the nets, whether they are impregnated with insecticide, the number of nets being used in the village and a variety of sociological factors that determine actual use of the nets in practice. The much lower protective efficacy of unimpregnated bed-nets is variable and depends very much on the way in which they are used (Do they have holes? Are they tucked under the mattress?, etc.). These considerations are relatively unimportant for ITNs. Many ITN studies have been conducted and these give an overall estimated all-cause child mortality reduction of 20% for their use in Africa. As a consequence many countries have taken up ITN programmes as an important component of their anti-malarial strategy. In recent years, the proportion of people who sleep under an ITN has risen substantially with increased sub-sidized deployment. Currently 289 million LLINs have been deployed in sub-Saharan Africa, enough to cover 76% of the 765 million people at risk of malaria. Impregnation of house-hold curtains, hammocks, clothing, or even cattle has been shown to reduce malaria. It has been assumed that ITNs work mainly through personal protection, but their mass insecticidal effect may be more important in some contexts. Thus the pro-tection afforded by sleeping without a net in a village where ITNs are used extensively, may be greater than sleeping under an ITN in a village where no-one else uses them.

Impregnated nets are effective throughout Africa but do not work in some areas (notably parts of South-east Asia), because of different human and mosquito behaviour. Obviously if malaria is contracted by vectors which bite in the early evening or early morning away from human habitation then ITNs are not going to be very effective.

REPELLENTS

Other simple preventive measures, including the application of permethrin or deltamethrin to clothing or the use of insect repellents such as diethyltoluamide (DEET) on exposed skin surfaces, are also effective and need not be prohibitively expen-sive. DEET is generally very safe, including in pregnancy. Coconut oil and DEET ‘soap bar’ preparations are available which are cheap, stable and readily applied. Houses can be

balance is also easier as renal failure is very unusual. However, the difficulties of providing adequate nursing in the tropics, of obtaining intravenous access and the small volumes of intrave-nous fluid required often mean that antimalarial drugs are given by the intramuscular or suppository routes. Children may deteriorate very rapidly in severe malaria. Sudden death is common in cerebral malaria but, if the child survives, recovery is more rapid than in adults. Iron deficiency is common in tropical countries, protects against malaria, yet commonly coexists with malaria. In general, the benefits of iron supple-mentation in iron deficiency, both on short-term anaemia and long-term neurocognitive development, outweigh the risks. Routine iron supplementation has not been recommended fol-lowing the results of a large carefully controlled study from Pemba, Tanzania in which the risks of death or severe illness of providing routine iron plus folic acid supplementation (in doses similar to those recommended by WHO) to young chil-dren exposed to high rates of malaria infection outweighed any immediate benefits.37 However these conclusions have been dis-puted and this remains a controversial area.

Malaria with Limited ResourcesMany patients with malaria are either untreated or treated inad-equately by self-medication. In many countries the private sector is the main source of antimalarial treatment. Fake or substandard drugs are common and incomplete treatment courses are often sold. Education of the public and the private commercial vendors is vitally important. Coherent and efficient schemes for the purchase and distribution of quality-assured drugs are needed. In order to slow the pace of antimalarial resistance it is essential that whoever gives antimalarial treat-ment (parent, relative, village health worker, shop assistant) ensures a full course of treatment is administered.

Most patients with severe malaria are not admitted to hos-pital; they are treated at home or at rural health clinics. Most deaths from malaria occur in or near home. Where intravenous infusions cannot be given, intramuscular administration is acceptable for quinine or the artemisinin derivatives. It is essen-tial that sterile technique is adhered to fully. Artesunate sup-positories are simple and effective alternatives to parenteral administration and as a pre-referral treatment they have been shown to reduce the mortality of children (under 5 years) with malaria who cannot take oral medication by 25%.Where injec-tions or suppositories are not possible then oral, or, if a tube is available, nasogastric instillation should be attempted, pending transfer of the patient.

Non-Pregnant Adults

Pregnant Women Children

Anaemia + ++ +++Convulsions + + +++Hypoglycaemia + +++ +++Jaundice +++ +++ +Renal failurea +++ +++ −Pulmonary oedema ++ +++ ±

aRequiring renal replacement. Elevated blood urea common in children with severe malaria.

TABLE 43.15

Relative Incidence of Severe Falciparum Malaria Complications

596 SECTION 9 Protozoan Infections

exposure to intermediate or high transmission (e.g. aircrews), travellers can be advised to carry a treatment course of antima-larial drugs with them. If they become ill and there are no medical facilities for malaria diagnosis and treatment, the treat-ment course is self-administered.

The use of antimalarial prophylaxis by the inhabitants of malarious areas remains controversial. It is generally agreed that pregnant women should take antimalarial prophylaxis if there is a significant risk of malaria, but that other adults should not. Chloroquine, pyrimethamine and proguanil are all considered safe in pregnancy, but are now largely ineffective against P. falciparum. Mefloquine is now considered safe. Tetra-cyclines and primaquine are contraindicated in pregnancy and atovaquone-proguanil has not been evaluated sufficiently. The use of antimalarial prophylaxis by children living in an endemic area has been shown to reduce mortality; in The Gambia administration of pyrimethamine and dapsone (Maloprim) in the 1–4-year age group reduced mortality by 25%. Despite the reduction in mortality, a reduction in the incidence of clinical attacks of malaria and anaemia, improved nutrition and, in older children, a decrease in absenteeism from school, this practice has not been generally adopted, largely because of concerns that widespread deployment of chemopro-phylaxis would encourage the spread of drug-resistant parasites and/or inhibit the development of naturally acquired immunity to malaria.

INTERMITTENT PRESUMPTIVE TREATMENT IN INFANCY (IPTI) AND CHILDHOOD (IPTC)

Following the success of IPT in pregnancy, the strategy of pro-viding a treatment dose of antimalarials to all infants in high transmission settings at the time of EPI immunizations (given

mosquito-proofed by using wire-mesh grilles over windows and designed in such a way as to discourage mosquito ingress. All these measures reduce the chances of an infection, but they do not eliminate it.

CHEMOPROPHYLAXIS

Although the early colonists devised many ingenious methods of taking quinine regularly (including ‘Indian tonic water’), they were generally neither pleasant nor fully effective. Quinine (a poor prophylactic) was relied upon by armies and colonists until after the Great War. The subsequent discovery of mepa-crine (quinacrine, atebrine) in 1934 gave the militaries an effi-cacious, albeit rather toxic, prophylactic which prevented malaria effectively during the Second World War. However, it was the introduction of chloroquine, the antimalarial bigua-nides and subsequently pyrimethamine after the war, that finally brought safe and effective antimalarial prophylaxis. The DHFR inhibitors (pyrimethamine, proguanil, chlorproguanil), primaquine and atovaquone all inhibit parasite development in the liver (pre-erythrocytic activity). They are sometimes called causal prophylactics. Chloroquine and mefloquine inhibit asexual blood-stage development but they do not prevent devel-opment of the liver stages. Thus the parasites emerge from the liver but cannot multiply in the red cells. Drugs with this action are called suppressive prophylactics. These drugs also have gametocytocidal activity against P. vivax, P. malariae and P. ovale, but not P. falciparum. Atovaquone–proguanil, doxycy-cline and primaquine have been added to the list of antimalarial prophylactics. Each is active against resistant P. falciparum but each must be taken daily. Antimalarial prophylaxis must be taken regularly to ensure therapeutic (i.e. suppressive) antima-larial concentrations are maintained. Recommendations vary considerably depending on risk, prevalence and drug resistance. Up-to-date recommendations are easily obtained on the inter-net (see, e.g.: http://www.who.int). Increasing drug resistance in recent years has meant that many prophylactic drugs can no longer be relied upon, particularly in areas of multiple drug resistance such as South-east Asia and South America.

The recommended prophylactic drug regimens are shown in Table 43.16. When prescribing antimalarial prophylaxis to trav-ellers, it is important to emphasize that no antimalarial is com-pletely effective and that a febrile illness could still be malaria. It is essential that prophylaxis is taken regularly and for most drugs continued for 4 weeks after leaving the transmission area. The need to take the drugs for a month after leaving the trans-mission area is to ‘catch’ any parasites acquired shortly before departure when they leave the liver. But drugs acting on the liver stages (atovaquone-proguanil, primaquine) can be stopped immediately. This is a particular advantage for travellers visiting a malarious area for a short time. It is prudent to begin prophy-laxis 1 week before departing for a malarious area so that toler-bility to the drug regimen can be assessed and therapeutic concentrations are present on arrival. In anglophone countries, chloroquine is prescribed weekly, but in francophone countries it is given once daily (this is theoretically preferable). Meflo-quine and pyrimethamine-dapsone are taken once a week and proguanil, atovaquone-proguanil, primaquine and doxycycline daily. Amodiaquine, quinine, sulfadoxine-pyrimethamine and the artemisinin drugs should not be used for prophylaxis.

In situations where the risk of infection is low, or there are no effective antimalarials available, or there is brief repeated

Weight Adjusted Dose for Children Adult Dose

CHLOROQUINE-SENSITIVE MALARIAChloroquineb 5 mg base/kg weekly or 300 mg base

1.6 mg base/kg daily 100 mg baseand/or

Proguanil 3.5 mg/kg daily 200 mg base

CHLOROQUINE-RESISTANT MALARIAMefloquinec 5 base/kg/weekly 250 mg base

orDoxycyclined 1.5 mg/kg daily 100 mg

orPrimaquine 0.5 mg base/kg daily

with food30 mg base

orAtovaquone-proguanil 4/1.6 mg/kg daily 250/100 mg

For current World Health Organization recommendations, see http://www.who.int.

aDetailed local knowledge of P. falciparum antimalarial drug susceptibility and malaria risk should always be obtained.

bChloroquine should not be taken by people with a history of seizures, generalized psoriasis or pruritus previously on chloroquine.

cMefloquine is not recommended for babies <3 months of age. Mefloquine should not be taken by people with psychiatric disorders, epilepsy, or those driving heavy vehicles, trains, aeroplanes, etc. or deep-sea diving.

dDoxycycline may cause photosensitivity. Use of sunscreens is recommended.

TABLE 43.16 Antimalarial Chemoprophylaxisa

43  Malaria 597

primaquine (0.5 mg base/kg) causes abdominal discomfort. Atovaquone-proguanil is remarkably well tolerated (similar adverse effects to proguanil alone) and highly effective.

Progress Towards a Malaria VaccineThere are no vaccines for parasitic diseases of humans. Despite considerable effort and expense (worldwide funding has run at US$70–100 million/year), a generally available and highly effec-tive malaria vaccine is still unlikely in the near future. The original goals of a vaccine producing sterile immunity (like the polio or yellow fever vaccines) without natural boosting are now considered unrealistic. The path of vaccine development has proved long and strewn with pitfalls, but there has been progress. Research has concentrated on all stages of the parasite life cycle: the sporozoite, the liver stage, the asexual blood stage and the gametocyte. Vaccines directed against the sporozoites and pre-erythrocytic liver stage are most advanced. The most effective vaccine produced to date was produced 40 years ago and consisted of irradiated sporozoites. This approach has been reactivated and an irradiated sporozoite vaccine is under devel-opment. Indeed there are numerous vaccines in various stages of development. By far the leading synthetic candidate, the result of 20 years development, is called RTS,S.39 The RTS,S/AS01 vaccine is a hybrid construct of the hepatitis B surface antigen fused with a recombinant antigen derived from part of the circumsporozoite protein (the protein coat of the sporozo-ite). Keys to the success of the vaccine are the immunogenic polymeric nature of RTS,S particles and the proprietary adju-vant AS01. In the first large double-blind efficacy trial con-ducted in Mozambique, about 2000 children one to 4 years of age were assigned to receive 3 doses of either RTS,S or a control vaccine. The primary end point was the time to the first episode of symptomatic P. falciparum malaria during a 6-month sur-veillance period; the vaccine’s efficacy in preventing clinical malaria was 29.9%. Of the 745 children in the RTS,S group 11 had at least one severe episode of malaria, compared with 26 of 745 children in the control group, a 58% protection against severe disease. However, the target population is younger chil-dren. In a large, multicentre phase 3 trial of RTS,S/AS01 15 460 children in two age categories: 6–12 weeks and 5–17 months – were enrolled. In the older group vaccine efficacy was 56% against all clinical malaria infections and 47% against severe malaria during a 12-month follow-up period. In the younger group vaccine efficacy against clinical malaria assessed in 6537 infants was 31% in the per-protocol population and efficacy against severe malaria was 26% in the intention-to-treat popu-lation.38 Decisions on whether to deploy this vaccine are expected in 2015. For the development of a blood-stage vaccine, work has concentrated on the different merozoite surface anti-gens (MSP1, MSP2, MSP3), the ring-infected erythrocyte surface antigen (RESA) and, to a lesser extent, proteins associ-ated with the rhoptries and the parasitophorous vacuole. Trans-mission blocking vaccines directed against P. falciparum gametocytes and vaccines against P. vivax sporozoite are also under development.

Chronic Complications of MalariaMalaria is a major cause of chronic ill health in the tropics, particularly in childhood. Repeated attacks of malaria cause anaemia, failure to thrive and probably also contribute

at 2, 3 and 9 months of age) was developed. The two drugs evaluated mainly have been SP and amodiaquine. The main benefit demonstrated has been a reduction in the incidence of clinical attacks of malaria and anaemia. The protection is for approximately once a month after administration so it is incomplete with current regimens. The evidence whether pro-tection extends to the second year of life as once claimed is conflicting. This is important as the majority of deaths occur after the first year of life. The benefits have been greatest in areas of high stable transmission. Predictably the benefits have been lower in areas of lower seasonal transmission where the major impact of malaria is after the first year of life, but studies in such areas (Senegal and Mali) showed that IPT given to older chil-dren during the malaria transmission season was remarkably effective in preventing malaria. Use of chemoprevention in older children is likely to be most effective in areas where a high level of malaria transmission is concentrated in a short period of the year. This has now been demonstrated conclusively and monthly administration of amodiaquine and sulphadoxine-pyrimethamine (seasonal malaria chemoprevention: SMC) is now recommended during the 3–4 rainy season months across the Sahel.38 It is estimated that in areas suitable for SMC, there are 39 million children under 5 years of age, who experience 33.7 million malaria episodes and 152 000 childhood deaths from malaria each year. Tens of thousands of deaths can there-fore be prevented using effective SMC. Obviously widescale use of prophylaxis, IPT or SMC will encourage the selection of resistance, although modelling studies to date are moderately reassuring. Another concern is that highly effective interven-tions such as prophylaxis together with ITN deployment might so reduce exposure that the acquisition of effective immunity was delayed, thereby increasing vulnerability to severe malaria at an older age. Again the available evidence is reassuring, but more information is needed on these important issues.

ADVERSE EFFECTS OF CHEMOPROPHYLAXIS

Adverse effects are a very important determinant of adherence to antimalarial prophylaxis regimens. As those taking the drugs prophylactically are healthy subjects, their tolerance of adverse effects is much lower than in treatment of malaria (where the patient often ascribes side-effects to the disease and takes the drugs only for a brief period). About 20% of patients taking prophylactic antimalarial drugs report some adverse effects. These are usually minor and do not require a change in prophylaxis. Nausea is the most common side effect. Chloro-quine causes pruritus in dark-skinned subjects. Dizziness, dysphoria and sleep disturbances are particularly associated with mefloquine, visual disturbances with chloroquine, and photosensitivity and monilia with doxycycline. The risks of neuropsychiatric reactions or seizures are approximately 1 : 10 000 and appear similar for mefloquine and chloroquine. There has been much televised publicity over the CNS adverse effects of mefloquine. Minor, but debilitating, CNS effects are reported more commonly in travellers taking mefloquine than in other groups of subjects. Mefloquine prophylaxis should not be offered to subjects with epilepsy, psychiatric disorders, or to subjects in whom any CNS disturbances could have disastrous consequences such as pilots, coach drivers etc. Primaquine (0.5 mg/kg per day) is well tolerated if taken with food. It should not be given to subjects who are G6PD deficient or who are pregnant. On an empty stomach

598 SECTION 9 Protozoan Infections

reported throughout the tropics. The highest incidence of hyper-reactive malarial splenomegaly (HMS) yet reported is in the Upper Watut Valley of Papua New Guinea, where 80% of adults and older children have large spleens. Genetic factors undoubtedly also play a role because within a malarious area the geographical distribution of HMS does not follow closely that of malaria transmission. In Ghana, 1st-degree relatives have a four times higher incidence of HMS than age- and location-matched controls.

PathologyThere is gross splenomegaly with normal architecture and lym-phocytic infiltration of the hepatic sinusoids with Kupffer cell hyperplasia. The massively enlarged spleen leads to hypersplen-ism with anaemia, leukopenia and thrombocytopenia. There is polyclonal hypergammaglobulinaemia with high serum con-centrations of IgM. High titres of malaria antibodies and a variety of autoantibodies (antinuclear factor, rheumatoid factor) are usually present. The hypergammaglobulinaemia is believed to result from polyclonal B-cell activation in the absence of adequate numbers of CD8+ suppressor T-cells, which have been removed by an antibody-dependent cytotoxic mechanism. Cell-mediated immune responses are otherwise normal. Immunoglobulin gene rearrangements have been dem-onstrated in a sub-group of patients with HMS. This indicates clonal lymphoproliferation and the potential for progression to malignant lymphoma or leukaemia.

Clinical FeaturesMost patients present with abdominal swelling and a dragging sensation in the abdomen. The malaria blood slide is usually negative. HMS commonly presents in pregnancy. The large, hard spleen is vulnerable to trauma. Acute left-sided abdominal pain suggests splenic infarction. The liver is also enlarged. Anaemia is often symptomatic and associated with pancytope-nia (hypersplenism) and there is an increased susceptibility to bacterial infections. The long-term prognosis of HMS is not good, with an increased mortality from infection. HMS appears to be a pre-malignant condition developing into lymphoma in some patients.

TreatmentThe enlarged spleen usually regresses over a period of months with effective antimalarial prophylaxis. Most experience has been gained with chloroquine and mefloquine. The liver also returns to normal and the IgM levels fall. Treatment is required for the duration of malaria exposure. Splenectomy is only rec-ommended if there is an unequivocal failure of prophylaxis given for at least 6 months and there is severe hypersplenism.

LymphomaIn some countries, Burkitt’s lymphoma is the most common malignancy of childhood. It is an uncontrolled proliferation of B lymphocytes and is associated with Epstein–Barr (EB) virus infections and malaria. The epidemiological association between malaria and Burkitt’s tumour is very strong. EB virus infections are widespread in the tropics and in most countries over 80% of children have serological evidence of infection by the age of 3 years. Normally, progression of EB virus in B lymphocytes is controlled by virus-specific cytotoxic T-cells (the atypical

to vulnerability to other infections and retard educational development. Chronic malaria is associated with certain specific syndromes.

QUARTAN NEPHROPATHY

The nephrotic syndrome, with albuminuria, hypoalbuminae-mia, oedema and variable renal impairment, is common in the tropics. Repeated or continuous P. malariae infection is associ-ated with childhood nephrotic syndrome in West Africa and Papua New Guinea. In the past, quartan nephropathy was also described in eastern Asia. It has disappeared from countries where P. malariae has been eradicated, such as Guyana, where Giglioli first described the relationship between malaria and nephrosis. This strong epidemiological association has been supported by pathological studies, although it is not known why certain individuals develop quartan nephropathy whereas the majority of those infected with P. malariae do not. The other species of malaria are also suspected of causing occasional glo-merulonephritis, but the evidence is less convincing than for P. malariae.

PathologyQuartan nephropathy is a chronic soluble immune complex nephropathy. Renal biopsy reveals a variety of abnormalities. There is commonly thickening of the subendothelial aspect of the basement membrane, giving rise to a double contour of argyrophilic fibrils. The changes are segmental initially. The capillary lumens narrow and become obliterated. On electron microscopy the basement membrane is irregularly thickened with lacunae of electron-dense material. Immunofluorescent study shows IgG and IgM along the capillary walls. In two-thirds of cases this is accompanied by C3 and other complement components. Coarse granular deposits with IgG3 are more common than fine granular or linear staining, which is more associated with IgG2 and a poor response to cytotoxic therapy. In acute disease P. malariae antigens are demonstrable in approximately one-third of cases, but these are not evident in long-standing nephrosis. The severity of the glomerulonephri-tis is usually graded: <30% glomeruli involved, grade I; 30–75% glomeruli involved + tubular atrophy, grade II; and >75% of glomeruli involved, with extensive tubular pathology, grade III. Very occasionally, adults develop a proliferative glomerulo-nephritis. This is not seen in children.

Clinical FeaturesThe pattern of renal involvement varies from asymptomatic proteinuria to full-blown nephrotic syndrome. Oedema, ascites or pleural effusions are usual presenting features. Anaemia and hepatosplenomegaly are common and many patients have fever on admission. The blood pressure is usually normal; the urinary sediment may show granular or hyaline casts in addition to proteinuria, but haematuria or red cell casts are rare. The disease usually progresses inexorably to renal failure over 3–5 years. Spontaneous remission is rare. Antimalarial treatment does not prevent progression and corticosteroids are usually ineffective. Some cases respond to cytotoxic therapy.

HYPER-REACTIVE MALARIAL SPLENOMEGALY

This is also known as the tropical splenomegaly syndrome. It occurs where transmission of malaria is intense and has been

43  Malaria 599

effective but expensive and may also reduce ventilation. Where domestic species of anophelines exist (e.g. A. stephensi in India), water jars, tanks or containers should be closed to prevent mos-quito access.

Simple measures such as introducing polystyrene balls to float on top of well water may be remarkably effective. The use of mosquito-proof bed-nets prevents human–vector contact, but they are considerably more effective in preventing malaria when impregnated with insect repellents or insecticides. Pyre-throid insecticide (permethrin, deltamethrin)-impregnated nylon nets are best and now long-lasting nets have been devel-oped which retain activity for many years. Insecticide-treated durable wall linings are currently under evaluation.

IMAGOCIDES

Although chemical agents, such as the larvicide Paris green and pyrethrum insecticides, had been widely used for vector control before the Second World War, the discovery of 2,2-bis-(p-chlorophenyl)-1,1,1-trichloroethane (DDT), with excellent activity against the adult mosquito (imagocidal activity), was a major advance in malaria control. DDT had residual imagocidal activity, which pyrethrum did not. It could be sprayed on the interior of houses and would kill or deter mosquitoes for many months afterwards. DDT, along with two other chlorinated hydrocarbon residual insecticides, gamma benzene hexachlo-ride (γ-HCH) and dieldrin, were the principal weapons in the campaign to eradicate malaria and they had a tremendous impact on health and development in the tropics. Imagocides can be classified into three general categories.

Pyrethrins and PyrethroidsThe naturally occurring compounds are light-sensitive and unstable, but the synthetic pyrethroids (permethrin, deltame-thrin) are both highly toxic to mosquitoes and stable, giving good residual activity. A single point mutation (resulting in phenylalanine or serine for leucine at position 1014) in the gene encoding a voltage-gated sodium channel protein is associated with pyrethroid and DDT resistance. Known as the pyrethroid knock down resistance (kdr) mutation, it has been found at several different locations, but predominantly in A. gambiae in West and South Africa. The mechanisms implicated in pyre-throid resistance include metabolic resistance based on elevated levels of cytochrome P450 as well as mutations in the target site, the kdr mutations. Insecticide resistance is spreading and may represent a serious threat to the efficacy of impregnated bed-nets and vector control.

Chlorinated Hydrocarbons (DDT, γ-HCH, Dieldrin)These are widely used as water-dispersible powders which form an aqueous suspension suitable for spraying. Resistance, human toxicity and ecological concerns have restricted the use of DDT in recent years. This valuable insecticide was vastly overused in the agricultural sector. Use in disease control was relatively small in comparison. But a global ban threw the baby out with the bathwater and threatened disease control in some areas where DDT was the only affordable and effective insecticide. Fortunately, the ban has been relaxed for vector-borne disease control. Used appropriately, DDT is still a very valuable malaria control tool (e.g. in Kwazulu Natal where pyrethroid resistant but DDT sensitive A. funestus caused an

mononuclear cells of infectious mononucleosis). This EB virus cytotoxic T-cell response is decreased significantly during acute malaria and there is increased proliferation of EB virus-infected lymphocytes. This may predispose to malignant transforma-tion. In areas of high stable transmission there is attenuated immune responsivity to EB virus in children between 5 and 9 years of age – the range of peak Burkitt’s lymphoma incidence.

In Ghana prospective studies of HMS and splenic lymphoma with villous lymphocytes suggest that a proportion of patients with HMS develop lymphoma. The prognosis is poor.

Malaria ControlIn his classic work on the prevention of malaria, Ronald Ross (1910) noted that in approximately 550 bc, Empedocles rid the Sicilian town of Selinus from a pestilence by draining the nearby marshes. Hippocrates (400 bc) knew that stagnant water and marshlands were unhealthy and that people living nearby would have enlarged spleens. The principles of drainage and landfill to control disease have continued since Roman times. The early attempts at joining the Atlantic and Pacific oceans were thwarted by disease, of which malaria was a major contributor, but during the final building of the Panama Canal, malaria was almost eradicated from the Canal zone by a vigor-ous combination of felling, drainage, house screening, pesticide use and antimalarial drugs (quinine). In recent years the prac-tices of vector control have evolved and environmental manage-ment and modification have come to the fore, both for disease control and for agricultural and other economic purposes. This is a complex and multidisciplinary field. Only a brief outline of the various approaches to malaria control will be described here. There are three main arms to malaria control; vector control through use of insecticides, deployment of insecticide treated bed-nets (or other treated materials) and use of effective drugs.

WATER-LEVEL MANAGEMENT

The oldest method of vector control – drainage – remains the most cost-effective, particularly in relatively dry areas where there is a high ratio of population to standing water. The practi-cal aspects of drainage are beyond the scope of this book. Water-level management to flush out mosquito breeding areas and to provide a hostile aquatic environment for mosquito egg and larval development, is an alternative to drainage. Changing water salinity or allowing organic matter pollution may also reduce vector populations. As always, major alterations to the environment should not be undertaken lightly: short-term ben-efits may be offset by long-term problems.

HUMAN BEHAVIOUR

Mosquitoes cannot fly far; most anophelines cannot fly more than 4 km and in general they remain within 2 km of their breeding sites. Of course they can be blown further and occa-sionally they take plane journeys and deliver malaria around airports in other countries. If humans do not live near breeding sites, the chances of infection are reduced. Many vectors bite inside houses and the design and protection offered by the dwelling are important determinants of malaria risk. Wire-mesh screens and other mosquito-proofing measures are

600 SECTION 9 Protozoan Infections

LARVICIDING

With the problems besetting use of residual imagocidal insec-ticides, there has been renewed interest in methods of larval control in recent years. These include environmental and water manipulation to prevent creation of mosquito breeding sites, the use of larvivorous fish and bacterial toxins and the applica-tion of chemical agents. Mineral oils were the first larvicides to be employed and diesel oil is still used today. Many of the ima-gocidal compounds described above are also used for larvicides. However, the organochlorines were highly effective but are no longer recommended because of their adverse environmental impact and the development of resistance. The organophos-phorus compounds are used widely and are relatively safe; for example, compounds such as temephos are safe to warm-blooded animals and fish and can be used to treat potable water.

OVERALL APPROACH

The objectives of a malaria control programme will depend on the prevailing epidemiological situation, the availability of resources and feasibility. One size definitely does not fit all! The first priority is the reduction of malaria mortality by making available facilities, personnel, diagnostics and drugs for diagno-sis and effective treatment. Then activities should focus on reducing malaria morbidity (such programmes should focus on malaria in childhood and malaria in pregnancy) and rely on use of effective drugs and vector control. In low transmission set-tings epidemics need to be anticipated. Having ‘secured’ the situation, it is also necessary to secure those areas free from malaria to prevent re-establishment of the infection. Finally and in a carefully planned and multifaceted programme, work to eliminate the disease should begin.

The Millennium Development Goals set a target of reducing the mortality of children under 5 years of age by two-thirds and halting and reversing the spread of malaria, by the end of 2015. Substantial resources have been made available to tropical countries for malaria control through the Global Fund for AIDS, TB and Malaria and other national and international donor agencies. This has resulted in substantial reductions in malaria morbidity and mortality. Estimated mortality has fallen by about one third over the past decade. The current global economic downturn and the emergence of resistance to insec-ticides and the artemisinin drugs represent a serious threat to these achievements.

epidemic in the late 1990s which was terminated by combined insecticide (DDT) spraying and artemether-lumefantrine deployment. Dieldrin is now considered too toxic to humans and it is no longer used.

AnticholinesterasesThese comprise the organophosphorus compounds (mala-thion, fenitrothion) and the carbamates (propoxur, trimetha-carb, bendiocarb). Although resistance to the organophosphates has limited use in some areas, these compounds are still distrib-uted widely. Malathion is the cheapest and most widely used. The anticholinesterases pose a potential health hazard to spray-ing teams, despite their wide therapeutic ratios.

General PrinciplesImagocides are also classified either by their portal of entry to the body of the mosquito, or to the method of application. Residual insecticides are applied as a deposit on to surfaces where the mosquitoes will rest (e.g. walls, ceilings). Space sprays fill the air with a mist or fog of insecticide. The choice of insec-ticide and application method will be determined by the sensi-tivity and behaviour of the local vectors and the nature of the environment. The anopheline mosquito vectors have countered these chemical assaults by changing their behaviour (resting and feeding preferences) and evolving resistance to the insecti-cides. This has had drastic consequences: reduced effectiveness; the necessity for more expensive replacements (to which resis-tance has also developed in some species); a disinclination of the chemical industry to invest further in a difficult and often unprofitable field; and as a consequence an inability of impe-cunious governments to pay for the new insecticides. Over 50 vector species are resistant to one or more of the organochlorine insecticides, over ten are resistant to the organophosphates and pyrethroid resistance is spreading. Most important, A. gambiae s. l., the dominant vector in Africa, has developed resistance to organochlorine insecticides in many areas. In Central America, A. albimanus has developed multiple insecticide resistance. In India the major vectors, A. culicifacies and A. stephensi, have become resistant to the organochlorines and malathion. Indoor residual spraying (IRS) is a major component of malaria control where anopheline vectors are susceptible. The number of people protected by IRS in the Africa region increased from 13 million in 2005 to 75 million in 2009, representing approximately 10% of the population at risk.

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