Information

First infection of malaria


As I know Plasmodium falciperum survive in either a host animal, human or mosquitoes.

But how does malaria come to infect either of them initially? I am interested in knowing evolution of malaria. How it infected first mosquitoes when neither human nor any other mosquito was infected?


First report of natural Wolbachia infection in wild Anopheles funestus population in Senegal

Until very recently, Anopheles were considered naturally unable to host Wolbachia, an intracellular bacterium regarded as a potential biological control tool. Their detection in field populations of Anopheles gambiae sensu lato, suggests that they may also be present in many more anopheline species than previously thought.

Results

Here, is reported the first discovery of natural Wolbachia infections in Anopheles funestus populations from Senegal, the second main malaria vector in Africa. Molecular phylogeny analysis based on the 16S rRNA gene revealed at least two Wolbachia genotypes which were named wAnfu-A and wAnfu-B, according to their close relatedness to the A and B supergroups. Furthermore, both wAnfu genotypes displayed high proximity with wAnga sequences previously described from the An. gambiae complex, with only few nucleotide differences. However, the low prevalence of infection, together with the difficulties encountered for detection, whatever method used, highlights the need to develop an effective and sensitive Wolbachia screening method dedicated to anopheline.

Conclusions

The discovery of natural Wolbachia infection in An. funestus, another major malaria vector, may overcome the main limitation of using a Wolbachia-based approach to control malaria through population suppression and/or replacement.


National Science Foundation - Where Discoveries Begin


Humans contract the malaria parasite when bitten by a female anopheles mosquito, like this one.


July 23, 2015

Malaria, one of the world's deadliest diseases, kills hundreds of thousands of people every year, spreading through a devastatingly effective cycle of incubation and transmission.

It starts when a female anopheles mosquito infected with a malaria parasite bites someone, injecting the single-celled parasite into the victim's blood. The parasite invades that human's liver cells, maturing, multiplying and moving into the bloodstream, making that person sick. When other mosquitoes bite that person, they ingest the parasite, which infects them. About 10 days later, when an infected mosquito happens upon uninfected humans, the whole cycle begins again.

It's a cycle that occurs throughout the year in densely populated areas, leaving nearly half the people in the world vulnerable to infection. Roughly 200 million people contracted malaria in 2013, according to the World Health Organization. Nearly 600,000 of them died--mostly children in Africa.

But what if there were a way to break that cycle?

Research by Jun Li, a University of Oklahoma biochemistry professor, could pave the way for a method to do just that. Through his work studying genetic changes in natural populations of mosquitoes, supported by the National Science Foundation's (NSF) Biological Sciences Directorate, Li has been able to isolate a mosquito protein critical to the malaria parasite's life cycle. That finding could one day lead to a treatment that doesn't affect just individuals, but entire communities by blocking the transmission of the disease.

"You won't be a source of malaria," Li said. "Think about this in the context of in a village, a city or the whole world. Your one shot could actually contribute to the reduction, or even elimination, of malaria."

Li, whose work is supported by NSF, the National Institutes of Health and the Oklahoma Center for the Advancement of Science, has had plenty of opportunities to think about the effects malaria can have on a community. Malaria's a fact of daily life in some of the most heavily populated areas of the world outside of the United States and Europe, he notes.

Those areas include his home in China--and Li knows well what malaria can do to a person.

"I suffered from malaria when I was a kid," he said. "When I was sick, I had almost no energy to stand. When I graduated with a Ph.D. and had a chance to study malaria, it was inspiring. It was a chance to work against this deadly disease."

A Critical Protein

Li's research sought to clarify a critically important question about malaria: How does the parasite interact with the host mosquitoes carrying it? What he found was a protein that serves an important function in malaria's lifespan--it allows the parasite to invade the insects' bodies.

"The mosquito is an organism. The parasite is also a pathogen," Li said. "Of course, when the parasite enters the mosquito by ingestion, there should be mechanisms through which the mosquito's system tries to prevent infection. But this protein actually acts like an anchor to help the parasite penetrate the physical barrier in mosquito guts."

He and his collaborators found the protein by collecting data from mosquitoes in Africa, along with blood from people infected with malaria. The researchers then fed that blood to mosquitoes, analyzing the rate at which the parasite multiplied in their bodies while also examining the insects' genotypes.

They found a particular protein, known as FREP1, in the part of mosquitoes' digestive systems known as the "midgut" makes the insects more susceptible to invasion by the parasite. Once the parasite gets into a mosquito, it multiplies.

"It is very important to note that the malaria has to infect the mosquito first before they can infect humans," Li said. "This protein is what allows that to happen."

Identifying that protein is a big step for Li and his collaborators. Now that they know the role it plays in spreading malaria, they can work on ways to use it to block transmission of the disease. Li says that such a treatment could involve injecting people with the mosquito protein. That patient would then generate an antibody against the protein. Any mosquito that bit the patient afterward would ingest the antibody--which would prevent the malaria parasite from taking hold in the mosquito's body.

"Your body becomes a dead end for the parasite," Li said, "and the community is protected."

Li is now collecting more data in Africa to support his work, and verifying that the protein plays the same role in multiple strains of malaria. He's hoping his research could help halt the transmission of a disease that he has a personal stake in stopping.

"When I'm working on this research, it's not just because it's my scientific interest," he said. "It actually will help keep millions of people healthy."

-- Robert J. Margetta, (703) 292-8070 [email protected]


University of Oklahoma biochemistry professor Jun Li collected data from mosquitoes in Africa.
Credit and Larger Version

The Centers for Disease Control and Prevention has mapped out malaria's two-host life cycle.
Credit and Larger Version

Mosquitoes ingest malaria during human blood meals in its gametocyte form, seen here.
Credit and Larger Version

A malaria parasite undergoes exflagellation, which normally occurs in the mid-gut of a mosquito.
Credit and Larger Version

Investigators
Jun Li

Related Institutions/Organizations
University of Oklahoma Norman Campus

Total Grants
$391,716

Related Agencies
National Institutes of Health
Oklahoma Center for the Advancement of Science


University of Oklahoma biochemistry professor Jun Li collected data from mosquitoes in Africa.
Credit and Larger Version

The Centers for Disease Control and Prevention has mapped out malaria's two-host life cycle.
Credit and Larger Version

Mosquitoes ingest malaria during human blood meals in its gametocyte form, seen here.
Credit and Larger Version

A malaria parasite undergoes exflagellation, which normally occurs in the mid-gut of a mosquito.
Credit and Larger Version


Cancer–malaria: hidden connections

Cancer and malaria exemplify two maladies historically assigned to separated research spaces. Cancer, on the one hand, ranks among the top priorities in the research agenda of developed countries. Its rise is mostly explained by the ageing of these populations and linked to environment and lifestyle. Malaria, on the other hand, represents a major health burden for developing countries in the Southern Hemisphere. These two diseases also belong to separate fields of medicine: non-communicable diseases for cancer and communicable diseases for malaria.

Despite the historical divide between cancer and malaria research, evidence accumulated over the past decade points to the need for understanding how the two diseases might influence each other biologically given their evolutionary history and epidemiology. In terms of evolutionary history, exposure of human populations to malaria, especially in Africa, is known to have shaped genetic variation at several loci in the human genome [1]. It is conceivable that if some of the genetic loci under previous or current selection by malaria are also involved in cancer, then this could impact the biology and epidemiology of both diseases. For example, long-term exposure to malaria infection in African populations led to selection of a variant of the Duffy antigen protein associated with reduced susceptibility to malaria [2,3]. While this gene variant evolved to confer protection against malaria, recent evidence suggests that it also influences inflammatory cytokines that are implicated in several cancers [4]. Consequently, this gene variant selected for malaria could also impact cancer outcomes in individuals of African ancestry, including African Americans who are not currently exposed to malaria [4]. Thus, existence of genes that play roles in both diseases could have significance in their biology and epidemiology. With respect to possible epidemiological associations between malaria and cancer, previous studies indicate that the prevalence of malaria is correlated with that of endemic Burkitt lymphoma [5] but negatively correlated with all-cause mortality across multiple cancers [6]. Today, the burden of malaria is decreasing in several countries where the disease has been endemic, while cancer cases are rising in many of those regions, specifically in Sub-Saharan Africa [7]. Here, we give four examples of biological mechanisms with independent studies demonstrating their important roles in both cancer and malaria, and highlight the role of these mechanisms in distinct developmental stages of the malaria parasite. We also discuss how they could impact the clinical management of the two diseases, not only in places where the two diseases co-occur but potentially in all world populations.

The first area where cancer–malaria interactions have been reported is in the human liver. The life cycle of the malaria parasite—Plasmodium—involves developmental stages in both the human host and the mosquito vector. In the human host, infection occurs through the bite of female anopheles mosquitoes that inject malaria parasite sporozoites into the human bloodstream. The sporozoites end up in the human liver, where they bind to cell surface proteins of hepatocyte cells, thereby infecting them. In a recent study, P53—the most highly mutated gene across several cancers—was shown to play a crucial role in the infection of hepatocytes by malaria parasite sporozoites. Specifically, Kaushansky et al. [8] demonstrated that mice expressing increased levels of p53 had low liver-stage infection by P. yoelii while those in which the gene was knocked out experienced a higher parasite burden in the liver. Furthermore, p53 agonists were more recently shown to eliminate Plasmodium liver stage infection in a mouse malaria model [9]. While the role of p53s in cancer as an anti-apoptotic protein is widely known, its involvement in malaria highlights the potential for leveraging the results from the vast research on this protein's role in cancer for the discovery of innovative drug targets in malaria. Such research effort would have to start with programmes aiming at deciphering the role of p53 in human malaria, as initial studies demonstrating its role in the disease have been in rodent malaria [8,9].

The second area where cancer–malaria interactions occur is in the blood stream. Hepatocyte development of malaria parasites takes about 2–10 days in most malaria species, during which infected individuals remain asymptomatic. During this period, sporozoites undergo multiple cell divisions eventually resulting in merozoites, which are released into the bloodstream through the rupture of hepatocytes. Merozoites then infect red blood cells (RBCs) by binding to a number of cell surface proteins and sugars exposed on the outer membrane of these cells. Among these cell surface receptors is the Duffy Antigen Receptor for Chemokines (DARC), which is used by the P. vivax malaria species to invade RBCs [2]. The role of this receptor in malaria parasite invasion was first demonstrated by the discovery that many African populations are resistant to infection by this malaria parasite species. It was subsequently discovered that this resistance was due to an inherited polymorphism in the promoter of the DARC gene that disrupts its expression specifically in RBCs but leaves its activity intact in other cell types [2]. This explains the low levels of P. vivax malaria in Africans when compared with Southeast Asia, where both P. vivax and P. falciparum infections are common. In addition to its role in malaria, DARC is a decoy chemokine receptor binding both C-C and C-X-C chemokines, but it lacks the capacity to couple to G-proteins, thereby failing to elicit immunological reactions [10]. Consequently, DARC can sequester chemokines in circulation and in the process dampen immunological responses. DARC has been demonstrated to be important in cancer in at least two ways. First, the ability of DARC to sequester chemokines could lower the concentrations of chemokines required for cancer metastasis and tumour neovascularization [11,12]. Indeed, increased expression of DARC in breast cancer cell lines was associated with the inhibition of tumour angiogenesis [11]. Second, DARC interacts with the tumour suppressor protein KAI1 (CD82), leading to the inhibition of proliferation and increased senescence of tumour cells [12]. Recently, the interaction between DARC and KAI1 on macrophages was shown to play a role in the maintenance of dormancy of long-term hematopoietic stem cells, providing an additional mechanism through which DARC may be important in cancer [13]. Therefore, lack of expression of this cell surface receptor on RBCs provides an evolutionary mechanism of protection against malaria infection at the price of having a broader impact on cancers.

Another area where cancer and malaria biology intersect is in relation to immune checkpoint molecules. In cancer, the expression of immune checkpoint molecules presents one of the key mechanisms allowing immune escape underlying the development of tumours. Among these molecules, PD-L1, expressed on the surface of tumours and antigen-presenting cells, interacts with PD-1, expressed on the surface of lymphocyte T cells, and this interaction eventually sends a negative signal to T cells. Over the past few years, public and private research programmes aiming at identifying and developing drugs targeting these immune checkpoint molecules have led to clinical developments and approval of breakthrough anti-cancer immunotherapies. Strikingly, Butler et al. [14] recently described PD-1 expression in T cells from children in Mali infected with P. falciparum, also suggesting T-cell exhaustion in human malaria. Moreover, they showed that blockade of PD-L1 and LAG-3—another intensively investigated immune checkpoint molecule—restored T-cell function and cleared blood-stage malaria in mice infected with P. yoelii [14]. These results thus open an exciting perspective on research on the investigation of anti-cancer immunotherapies in malaria patients, as well as on the exploration of immune checkpoints' gene variants in malaria-endemic regions—where human populations may have been selected for different expression profiles of these genes to resist malaria infection.

The connection between malaria and cancer is also demonstrated in the biology of placental malaria. Malaria in pregnancy is associated with serious complications including low birth weight, stillbirth and spontaneous abortion [15]. While malaria-infected pregnant women may still exhibit the common malaria symptoms, some may be asymptomatic or present with milder symptoms [16]. Malaria parasites sequester in the placenta by using its variant surface antigen (VAR2CSA) to adhere to the placenta [17,18]. VAR2CSA contains six Duffy binding-like domains that bind to chondroitin sulphate A (CSA), a glycosaminoglycan expressed on the placental surface [18]. Interestingly, CSA is also present on the surface of several malignant cells where it is linked to proteoglycans including CD44 and CSPG4 [19,20]. In tumours, CSA enhances aggressiveness and metastatic capacity of malignant cells [19]. The ability of VAR2CSA to specifically bind to the placental form of CSA modification on cancer cells makes it an attractive guide for targeting cancer cells [21]. In line with this, recombinant VAR2CSA (rVAR2CSA) was shown to bind CSA present on the surface of tumours. Subsequently, fusion of diphtheria toxin or conjugation of hemiasterlin derivatives to VAR2CSA inhibits tumour growth and metastasis in vivo [21,22]. These results demonstrate how insight gained on a protein that an infectious agent uses to target human cells can be leveraged to target a non-communicable and devastating disease—cancer—and potentially benefit human health.

It is important to note that the unexpected connections between malaria and cancer are not unique to the two diseases but are among those across several diseases at the molecular and epidemiological levels. At the molecular level, it is imperative that as humans have a finite number of genes, many of which are pleiotropic, distinct diseases are bound to interact with the same set of genes. Furthermore, many diseases may perturb similar metabolic and/or immunological processes leading to dependencies between the distinct diseases. At the epidemiological level, disease comorbidities may also arise due to shared environmental risk factors. In this commentary, we focus on malaria and cancer as the two diseases are commonly viewed as fundamentally distinct: whereas malaria is an infectious disease, cancer is non-communicable. It is rare to find collaboration between researchers in the two disease areas or researchers working on the two diseases simultaneously.

The examples provided in this commentary are by no means exhaustive of the connections that may exist between malaria and cancer. However, we have chosen these examples as illustrative for a number of reasons: (i) the connections between cancer and malaria presented here would not have been easily predicted and were discovered serendipitously, (ii) these connections cover distinct developmental stages of the malaria parasite, and (iii) the connections between cancer and malaria considered here show how insights from cancer can be used to find new ways of combatting malaria and vice versa.

The historical divisions between research on cancer and malaria might therefore reflect intellectual constructions rather than biomedical realities. Such artificial silos might negatively impact the way we tackle these two diseases that affect more and more patients worldwide. At the medical level, it might already bias the management of patients affected by malaria and cancer, an increasing problem in developing countries. Hidden links between malaria and cancer also point to potential inefficiencies in the organization of research on diseases. Only a small subset of research projects proactively focus on several types of diseases simultaneously. When it comes to research funding, it is even harder to defend these types of projects in front of funders that generally require that the projects they support only focus on one disease or one disease family. Such organization of care, research and funding might ultimately prevent cross-fertilization in medicine. It makes it even harder for the public to apprehend the high degree of interconnections between human diseases and for researchers to leverage insights across several diseases for the advancement of clinical medicine.


Pre-Erythrocytic (PE) Plasmodium Infection: Biology of Transmission - The Skin and Liver Stages

The sporozoite and liver stages comprise the PE stage of infection. Unlike the BSs, the PE stages are asymptomatic, are small in numbers during natural infection, and are not as antigenically variant. These characteristics render them extremely attractive targets for malaria intervention (13). Dissimilar to the BSs, the PE stages of P. falciparum cannot be easily generated in the laboratory and although some aspects of human infection can be modeled in vitro, these systems have limitations. Therefore, most of our knowledge on PE biology has been derived from studies of rodent malaria parasites, which were originally isolated from wild African rodents and subsequently adapted to laboratory mice (14�). For transmission research, the biology of skin infection between different rodent parasite species (Plasmodium yoelii and Plasmodium berghei) made it attractive to speculate that parasite behavior in the skin should be similar in Plasmodium species that infected humans. However, the recent identification of the development of exo-erythrocytic merozoites in the skin of P. berghei-infected mice (17) but not in P. yoelii-infected mice (18) identified one point of divergence even within these two closely allied parasite species. Thus, whether the development of skin exo-erythrocytic merozoites occurs in human Plasmodium parasites remains an open question, as preventive therapeutics targeted to the liver stage might be ineffective in the skin. Given that this skin stage of infection can only be adequately modeled within the three-dimensional architecture of the skin tissue, recent advances in the engraftment of human skin into immunodeficient mice (19) represent an exciting opportunity to examine interaction of human parasites with human skin components in vivo and an opportunity to explore the occurrence and relevance of “skin exo-erythrocytic merozoites” in Plasmodium parasites that infect humans.

Each sporozoite that reaches the liver invades a single hepatocyte, transforms into a trophic stage, and then commences liver stage development (also called exo-erythrocytic development). Traditional rodent malaria models have been critical to the iden-tification of host hepatocyte surface factors necessary for sporozoite invasion (7, 20). Recently, human liver-chimeric mice have been employed to examine the contribution of these invasion factors in human malaria parasite liver infection (21). The efficient engraftment of huHeps into mice is dependent on an environment of severe immunodeficiency to limit huHep rejection coupled with the elimination of mouse hepatocytes to provide the huHep a niche in the liver parenchyma. Three different mouse models have been utilized for huHep engraftment and have been used to assess liver stage infection by Plasmodium parasites infecting humans. The SCID Alb-uPA model expresses the toxic urokinase plasminogen activator (uPA) under the control of an albumin promoter in the livers of the highly immunodeficient Severe Combined Immune Deficiency mice (SCID Alb-uPA). Upon engraftment with huHeps, these mice become susceptible to infection with P. falciparum sporozoites and support complete liver stage development including the release of exo-erythrocytic merozoites that egress and invade human RBCs (huRBCs) ex vivo (22, 23). An alternate to the induction of hepatotoxicity by uPA transgene expression is genetic ablation of fumarylacetoacetate hydrolase (FAH) in mice resulting in acute liver failure which can be rescued by administration of the drug, 2-(2-nitro-4-fluoromethlbenzoyl)-1,-3-cyclohexanedione (NTBC). Crossing these FAH −/− mice onto the severely immunocompromised C57BL/6 Rag2 −/− IL2rγ −/− mouse generated FAH −/− Rag2 −/− IL2rg −/− (FRG) mice. These mice have also been backcrossed onto the non-obese diabetic (NOD) background (FRGN), which additionally renders them hospitable to transplantation with CD34 + hematopoietic stem cells (HSCs) (24). Using NTBC cycling during engraftment, these mice can exhibit over 90% engraftment with huHeps (FRG huHep) (25), are susceptible to infection with both P. falciparum and P. vivax sporozoites (26, 27) and support full liver stage development, including the release of exo-erythrocytic merozoites capable of invading huRBCs that were infused into the mice. When infected with P. vivax, FRG huHep mice also harbor non-replicating hypnozoites (27). More recently, the TK-NOG (NOD/Shi-scid/IL2rg −/− ) mouse has been developed as yet another mo-del for P. falciparum PE infection (28). These mice express the herpes simplex virus thymidine kinase transgene under the control of the albumin promoter on the NOD SCID IL2rγ −/− background. Destruction of mouse hepatocytes is achieved by treatment with ganciclovir, allowing repopulation of the liver with huHeps. TK-NOG mice support P. falciparum and Plasmodium ovale sporozoite infection and liver stage development (28).


Malaria: Obstacles and Opportunities (1991)

For centuries, malaria parasites have successfully evaded the biological defenses of their human hosts. Researchers are perplexed by the complexity of these organisms, and many questions remain unanswered. By the year 2010, advances in the field of parasite biology will have exposed many of the complex biochemical mechanisms that allow this evasion to occur. A detailed understanding of how malaria parasites recognize and invade human liver and red blood cells, for example, how their multistage life cycle is regulated and how they rapidly become drug resistant will have provided a major boost to efforts to develop malaria vaccines and will have resulted in innovative approaches to more durable antimalarial drugs.

WHERE WE ARE TODAY

The Parasite

The human malaria parasite&mdashactually four species of the genus Plasmodium&mdashundergoes over a dozen distinguishable stages of development as it moves from the mosquito vector to the human host and back again. One way to conceptualize this complex life cycle is to consider it in three distinct parts: the liver phase, the blood phase, and the mosquito phase.

Depending on the developmental stage and species, malaria parasites can be spherical, ring shaped, elongated, or crescent shaped, and can range in size from 1 to 20 microns in diameter (1 micron equals 1 millionth of a meter or approximately 125,000 of an inch). By comparison, a normal red blood cell has a diameter of about 7 microns.

Although the four species of human malaria parasites are closely related, there are major differences among them. Plasmodium falciparum, the most pathogenic of the four species, has been found to be more closely related to avian and rodent species of Plasmodium than to the other primate and human species (McCutchan et al., 1984). The following sections of this chapter discuss general aspects of malaria parasite biology, with a focus on P. falciparum. Interspecies differences are noted where appropriate.

Parasite-Host Interactions
Liver Phase

The liver phase of malaria begins when the female anopheline mosquito injects the sporozoite stage of the parasite into the human host during a blood meal (see Chapter 2, Figure 2-3). After just a few minutes, the sporozoites arrive at the liver and invade the liver cells (hepatocytes). Over the course of 5 to 15 days, depending on the species, the sporozoites undergo a process of asexual reproduction (known as schizogony, the &ldquosplitting process &rdquo) that results in the production of as many as 30,000 &ldquodaughter&rdquo parasites, called merozoites. It is the merozoites that, once released from the liver, carry the malaria infection into the red blood cells (erythrocytes).

The surface of the sporozoite is coated with many copies of a protein that is thought to play a key role in host cell recognition and perhaps cell invasion. Antibodies to certain portions of this circumsporozoite protein can prevent sporozoites from entering liver cells in culture and may play an important role in protecting against infection. People exposed to irradiated sporozoites are protected against infection by unaltered sporozoites. This level of immunity has not yet been obtained with use of subunit, synthetic, or gene-cloned vaccines (see Chapter 9).

The circumsporozoite protein can be detected throughout the parasite 's development in the liver and may also be associated with liver-stage merozoites. The circumsporozoite protein has a unique structure of immunodominant, highly repetitive complexes of amino acids (Santoro et al., 1983). Although different Plasmodium species have distinct antigenic properties, the repetitive complexes seen in P. falciparum sporozoites are also seen in P. vivax and other parasite species, although the actual makeup of these proteins is species specific.

It is not clear how the parasite arrives at the surface of the hepatocytes, since liver cells are not in direct contact with the blood. Also, although many mechanisms have been postulated, details of how the parasites invade liver cells remain obscure (Miller, 1977). The physiological changes that the sporozoite undergoes during the shift from a cold-blooded mosquito host to a warm-blooded human host are not well understood, nor is it understood how the parasite manages the reverse situation, as the sexual stage precursor&mdashthe gametocyte&mdashtravels from human to mosquito. In the latter case, there is evidence suggesting that changes in temperature and pH help stimulate the emergence of the gametes in the insect midgut. Schizogony in the liver had been thought to proceed without pathological manifestations, but recent evidence points to the existence of inflammatory mechanisms and cellular infiltration, including cytotoxic T cells, all of which may be immunologically important.

It has been known for many years that the symptoms of malaria can recur without reexposure to the parasite, but the biological mechanisms underlying this phenomenon have only recently been determined. The reappearance of symptoms may either be due to relapse, i.e., the development of latent sporozoites, called hypnozoites, in the liver following a period during which the blood itself was parasite free, or recrudescence, a sudden upsurge of blood-stage parasites after a protracted period of very low parasite density (Krotoski et al., 1982). Relapses occur only in P. vivax and P. ovale infections, while recrudescence generally occurs in P. falciparum and P. malariae. Although the hypnozoite does not appear to undergo development during its dormant state, the fact that hypnozoites are readily eliminated by the action of the drug primaquine implies some level of metabolic activity.

Blood Phase

When merozoites are released from the liver into the bloodstream, asexual blood-stage reproduction, or erythrocytic schizogony, has begun. Parasite invasion of red blood cells unfolds in four steps: attachment of the merozoite to the erythrocyte, rapid deformation of the red blood cell, invagination of the erythrocyte membrane where the parasite is attached and subsequent envelopment of the merozoite, and the resealing of the erythrocyte membrane around the parasite (Aikawa et al., 1978 Hadley et al., 1986 Perkins, 1989 Bannister and Dluzewski, 1990 Wilson, 1990).

Attachment, an event separate from invasion, may occur without endocytosis. There is indirect evidence that organelles at the tip of the parasite, the apical complex, are involved in invasion. For example, only invasive stages have apical organelles, and the organelles rapidly disappear after invasion. Although little is known about the biochemical functions associated with these organelles, efforts to clone proteins found in them may

provide some insight into their roles (Coppel et al., 1987). The attachment phase is a potential target for vaccine developers, since antibody that interferes with this process may prevent parasite invasion of red blood cells. Red blood cells have receptors for malaria parasites on their surfaces, although these receptors may be different for different parasite species. The best example of a parasite receptor on a red blood cell is the Duffy antigen, which is recognized by P. vivax this parasite cannot invade Duffy-negative cells. Of considerable interest is the recent cloning and characterization of the P. vivax (and P. knowlesi) gene encoding the parasite protein which binds to the Duffy antigen (Adams et al., 1990 Fang et al., 1991). This protein is located in one of the apical organelles (micronemes) thought to play a role in the invasion process. Unfortunately, our understanding of the attachment process for P. falciparum is less sophisticated.

Although the exact mechanism of invasion is still unresolved (see reviews cited above), it is likely that specific parasite proteases are involved (Perkins, 1989). Suggested candidates include a neutral endopeptidase (Bernard and Schrevel, 1987 Braun-Breton et al., 1988) and a chymotrypsin-like enzyme (Dejkriengkraikhul and Wilairat, 1983) selective protease inhibitors could thus be of potential interest as chemotherapeutic agents which prevent invasion.

After invasion, the parasite lies within a membranous parasitophorus vacuole, where it synthesizes nucleic acids, proteins, lipids, mitochondria, and ribosomes and assembles these components into new merozoites (Ginsburg, 1990b). The entire erythrocytic asexual cycle takes between two and three days to run its course, depending on the species.

Once merozoite assembly is completed, the erythrocyte ruptures and merozoites are released into the plasma, where they attach to other erythrocytes and begin the process anew. Some merozoites, for reasons not well understood, differentiate into the sexual forms of the parasite, the gametocytes. The factors that determine the sex of the gametocyte are unknown. Gametocyte development takes between 2 days (for P. vivax) and 10 days (for P. falciparum). The release of merozoites precipitates malaria's classical paroxysms of fever, chills, headache, myalgia, and malaise. Although the cause of the fevers is unknown, one theory is that endotoxin-like substances may be released during schizont rupture. The paroxysm itself may be due to transient increases in cytokines, such as interleukin-1 and tumor necrosis factor (Kwiatkowski et al., 1990). Children living in highly endemic areas often have significant parasitemias without symptoms, leading researchers to suspect the presence of &ldquoantitoxic &rdquo immunity.

Mosquito Phase

When gametocytes are taken up during a mosquito's blood meal, a number of factors, including temperature, concentrations of oxygen and carbon

dioxide, pH, and a mosquito exflagellation factor, are thought to contribute to the maturation of gametocytes. Male microgametes are released during a process called exflagellation. Fusion of the female macrogamete with a single microgamete results in fertilization and the formation of the ookinete. The ookinete migrates to the wall of the mosquito midgut, where it penetrates the peritrophic membrane and epithelium and comes to rest on the external surface of the stomach. Over a period of days, this stage of the parasite matures into an oocyst containing up to 10,000 motile sporozoites. When the oocyst ruptures, the sporozoites enter the mosquito circulation and travel to the salivary glands, where they are injected into the human host when the mosquito feeds. The number of sporozoites that enter the human host during a single blood meal is thought to be highly variable.

Parasite Physiology and Biochemistry
Feeding

Malaria parasites feed by ingesting intact erythrocyte cytosol, the internal fluid portion of the cell, through an organelle, the cytostome. This process has been reconstructed recently in three dimensions from micrographs (Slomianny, 1990). When the cytostome closes around cytosol, it creates a membrane-bound vacuole. In P. falciparum, the ingested host cytosol is then exposed to a mixture of potent digestive enzymes. That digestion of hemoglobin is required for parasite survival was shown in experiments in which hemoglobin was chemically cross-linked, making it resistant to degradation by P. falciparum proteases and cathepsin D parasites which invaded red blood cells with cross-linked hemoglobin failed to develop to trophozoites and eventually died (Geary et al., 1983). Recently, two proteases important for hemoglobin digestion in P. falciparum have been characterized. One is a cysteinyl proteinase (Rosenthal et al., 1988) and the other, which apparently initiates digestion, is an aspartyl proteinase (Goldberg et al., 1991). Reversible inhibitors of the cysteinyl protease (such as leupeptin) block hemoglobin digestion and suspend parasite growth. However, growth resumes even after prolonged incubations when the inhibitor is removed. Irreversible protease inhibitors, on the other hand, killed the parasites (Rosenthal et al., 1988). Selective protease inhibitors which block either of these two enzymes would be of considerable interest as potential antimalarial drugs. As the process of hemoglobin digestion becomes better understood (Goldberg et al., 1990), additional sites for chemotherapeutic intervention should be uncovered.

During digestion, about three-fourths of the host cell hemoglobin is destroyed. The residue left from this process is an insoluble particulate complex call hemozoin this complex contains, among other material, the

heme derived from hemoglobin. Although the chemistry of this complex is becoming better understood (Goldie et al., 1990 Slater et al., 1991), its role in pathogenesis, drug action, or immunology remains undocumented.

Permeability Changes in Erythrocyte Membranes

Following parasite invasion, the intracellular metabolism of infected erythrocytes increases significantly. Nutrients must be brought in from outside, and waste products must be disposed of expeditiously. The cell membrane responds by increasing its capacity to transport a variety of substrates in and out of the erythrocyte, including essential amino acids, nucleosides, lactate, and fatty acids. The changes in membrane permeability allow a number of substrates, that otherwise would not be let in at all or would be let in to a limited degree, to enter the infected red blood cell. These substrates include hexitols, acidic and neutral amino acids, several small inorganic ions, and organic acids.

The appearance of new erythrocyte membrane transport pathways is the result of host cell &ldquoremodeling&rdquo by the intracellular malaria parasites. It is thought that in remodeling, proteins of parasite origin become associated with host membrane components, either by adhering to the inner aspects of the membrane or by inserting themselves directly into the membrane. The experimental data strongly support this hypothesis (Haldar et al., 1986 Ginsburg and Stein, 1987 Cabantchik, 1989, 1990 Ginsburg, 1990a Tanabe, 1990a,b).

Other Parasite-Directed Changes in Erythrocyte-Membrane Structure

In P. falciparum, the trophozoite stage inserts new molecules into the host erythrocyte plasma membrane. These new membrane components are responsible for the sequestration of mature parasite stages in capillaries by the process of cytoadherence. Although among the human malaria parasites only P. falciparum exhibits cytoadherence, P. vivax also induces alterations in the infected erythrocyte membrane. Ultrastructure studies have shown that the membranes of erythrocytes infected with P. vivax contain caveolar structures that appear to be connected to vesicles (Atkinson and Aikawa, 1990 Barnwell, 1990). These caveolae-vesicle complexes appear to play a role in parasite interaction with the extracellular environment. They induce antibody production and are antigenically highly variable.

Nutrition and Metabolism

A malaria infection initiated by a single malaria parasite may produce as many as 10 billion new organisms. Nearly all the metabolic processes of

the parasite are focused on supporting this enormous reproductive effort. The relatively recently acquired ability to cultivate P. falciparum in vitro has greatly expanded biochemists' ability to study parasite nutrition and metabolism.

In P. falciparum, glucose can be replaced by fructose, but the parasite will not develop in vitro when another sugar, such as galactose, mannose, maltose, or ribose is substituted (Geary et al., 1985a). Although malaria parasites are capable of synthesizing the amino acids glutamate, aspartate, alanine, and leucine from glucose, they probably acquire them either through digestion of hemoglobin or from sources outside the red blood cell. Most early studies on the uptake of amino acids by malaria parasites utilized the animal parasites P. berghei, P. lophurae, P. gallinaceum, and P. knowlesi either in vivo, an approach in which experimental parameters are difficult to control, or in vitro, using less than ideal culture procedures. Observations on parasite biochemistry using cultures of P. falciparum have not always supported conclusions drawn from these flawed models.

In experiments using cultured P. falciparum, it has been shown that 13 of the 20 amino acids can be obtained from the digested erythrocyte cytosol the parasite must receive the other seven amino acids from sources outside the erythrocyte. Selective transport facilities may exist for any or all of these amino acids in P. falciparum, but evidence from other Plasmodium species suggests that this is not the case for all amino acids. Attempts to supplement glutamine, one of the amino acids, with other metabolites have been unsuccessful. Both glutamate and glutamine are required for continuous cultivation, indicating that interconversion is limited at best.

There appears to be only one vitamin, calcium pantothenate, that is not provided by the erythrocyte but is needed by the parasite for survival. Evidence for this comes from in vitro studies using culture medium containing this vitamin (Divo et al., 1985a). The malaria parasite's requirement for para-aminobenzoic acid and folic acid is well documented. The requirement for these vitamins, found in red blood cells, is probably strain specific. Sulfonamides, which inhibit folic acid synthesis, have been used as antimalarial drugs for years. The story of folic acid metabolism is complicated, however, and not all sulfonamides are equally potent as antimalarials. Surprisingly, unlike most organisms, P. falciparum does not seem to require biotin. The ability to develop in the absence of this vitamin was demonstrated by growing the parasites in the presence of several biotin antagonists, including avidin (Geary et al., 1985b).

Pyrimidines and purines are the two main building blocks of DNA. Malaria parasites can synthesize the former de novo, but purines are a required nutrient (Gero and O'Sullivan, 1990). Hypoxanthine is the preferred purine source, but other purines readily substitute for hypoxanthine. Studies of the kinetics of DNA synthesis in P. falciparum have revealed

that incorporation of labeled purines into DNA begins approximately 30 hours after merozoite invasion and increases logarithmically for another 14 to 18 hours, when schizogony is completed.

Because erythrocytes contain considerable concentrations of amino acids and vitamins that may be important to parasite development, it is difficult to determine in experimental settings whether decreased parasite viability due to nutritional factors is a result of effects on the parasite itself or on the red blood cell. Thus, it is not known whether nutrients identified as crucial for malaria parasite cultivation are required for erythrocytes only, parasites only, or both. For example, the amino acids glutamine, glycine, and cysteine, while necessary for long-term survival of erythrocytes in culture, may not be required parasite nutrients.

Energy Transformations and Mitochondria

There has been considerable debate about whether the erythrocytic stages of mammalian malaria parasites possess mitochondria, the energy-producing organelles essential for all life forms. The falciparum parasite uses glucose as its primary energy source. In fact, glucose utilization is significantly greater in the infected erythrocyte than in the uninfected cell. Progress is being made in the characterization of the enzymes involved in glycolysis in P. falciparum (Roth et al., 1988 Roth, 1990). However, there is no evidence supporting the presence of a Krebs cycle, a key energy-producing process of the mitochondria.

The presence of mitochondria in the erythrocytic asexual stages of P. falciparum has recently been shown, but their actual function is not well understood (Divo et al., 1985b). Recent advances in the molecular biology of the mitochondrial DNA of malaria parasites may help to unravel the role of the mitochondrion (Gardner et al., 1988). The importance of this organelle to the parasite is underscored by the fact that mitochondrial toxins are highly lethal. Antibiotics used to treat falciparum infection, such as the tetracyclines, clindamycin, and erythromycin, appear to work by blocking the development of parasite mitochondria (Prapunwattana et al., 1988). Of great interest in this regard is the recent finding that mitochondrial DNA of P. falciparum encodes an RNA polymerase which is closely related to prokaryotic polymerases and is sensitive to rifampicin, potentially explaining the antimalarial activity of this drug (Gardner et al., 1991).

The erythrocytic stages of many mammalian malaria parasites appear not to derive their metabolic energy through classical electron transport. The mitochondria may participate in ion transport, but the role this plays in metabolism is unclear. It is not known whether components analogous to those present in the mammalian terminal electron transport system function in the malaria parasite, and for what purpose, since the organism, like many


Malaria Research Program Investigators

Dr. Wellems focuses on the determinants of drug resistance, immune evasion, and disease virulence in malaria. Areas of study include the antimalarial drug resistance and factors that affect clinical outcome after treatment, malaria protection conferred by human hemoglobinopathies and other red cell polymorphisms, antigenic variation by Plasmodium falciparum parasites, and molecular mechanisms of malaria parasite infectivity and pathogenesis. Research activities on the NIH campus are integrated with field studies in Africa and Southeast Asia.

Sanjay Desai
Apicomplexan Molecular Physiology Section

Dr. Desai’s group studies the molecular and cellular biology of malaria parasite adaptation to intracellular growth within erythrocytes. His studies have identified two unusual ion channels that play a central role in nutrient and ion transport between plasma and parasite compartments. One of these channels, the plasmodial surface anion channel (PSAC), is exposed on the infected erythrocyte surface and is being actively pursued as an antimalarial drug target.

Louis Miller
Malaria Cell Biology Section

Dr. Miller’s study of the pathogenesis of malaria includes research on the mechanism by which malaria parasites invade erythrocytes, including the study of parasite ligands and erythrocyte receptors the mechanism of antigenic variation the molecular basis for cerebral malaria and rosetting and the binding of var gene products to endothelium.

Xin-Zhuan Su
Malaria Functional Genomics Section

Dr. Su’s laboratory develops and uses genetic and genomic approaches to study host-malaria parasite interaction and molecular mechanisms of the interaction using the rodent malaria parasite Plasmodium yoelii as a model. He has characterized large numbers of microsatellites and single nucleotide polymorphisms (SNPs) from several P. yoelii parasites and performed various genetic crosses to identify parasite genes linked to parasite development, virulence, and drug resistance. He is studying host immune signaling pathways in response to parasite infections, focusing on innate signaling and regulation of type I interferon production and inflammatory responses after malaria infection. He is also interested in anti-malarial drug screening and mechanism of drug resistance.

Joel Vega Rodriguez
Molecular Parasitology and Entomology Unit

Dr. Vega-Rodriguez’s group, the Molecular Parasitology and Entomology Unit, LMVR/NIAID/NIH, studies the biology of the malaria parasite during sexual reproduction in the mosquito and during sporozoite transmission, by characterizing essential vector-parasite and host-parasite interactions. Areas of study include the role of vector and host factors for sporozoite infectivity, and molecular mechanisms required for Plasmodium sexual reproduction. The main goal is to identify new targets for malaria interventions including chemotherapy, vaccines, and transgenic mosquitoes. We use a combination of molecular, cellular, and entomological technologies including single-cell transcriptomics, proteomics, parasite and mosquito transgenesis, RNA interference, and microscopy.

Malaria Immunity and Pathogenesis

Patrick Duffy
Pathogenesis and Immunity Section

Dr. Duffy conducts human and animal studies of malaria pathogenesis and host immunity, including population-based studies in African communities exposed to Plasmodium falciparum, and controlled human infection studies. His natural history studies emphasize mechanisms of immunopathogenesis in pregnant women and children, as well as community-wide studies of malaria transmission, with large collaborative cohort studies in progress in Mali and elsewhere. His controlled human infection studies at the Clinical Center and in the field seek to understand immunity against preerythrocytic parasites and to model parasite transmission to mosquitoes. He directs the intramural malaria vaccine program at NIAID, which leads the world in clinical development of transmission-blocking vaccines, studies whole sporozoite vaccines in humans, and is designing vaccine candidates against placental malaria and severe childhood malaria.


Infection Outcomes

The outcomes that follow a malaria infection can vary from no symptoms to life-threatening disease and death. The precise reasons why people respond in different ways to the same parasite infection are still unknown, experts say.

Researchers from the University of Edinburgh, in collaboration with teams at the Universities of Oxford and Glasgow and the Wellcome Trust Sanger Institute, explored infection outcomes in 14 volunteers who were injected with malaria parasites.

Scientists studied how the volunteers responded to the parasites over the course of 10 days. The group were then treated with antimalarial drugs to cure the infection before there was any risk of them developing severe symptoms.


A Guide to Malaria

Malaria is one of the most ubiquitous diseases known--there are more than 125 different species of malaria that infect mammals, birds and reptiles, which indicates an early origin. It has probably afflicted humans throughout our evolutionary history, although the first historical reports of symptoms that match those of malaria date back to the ancient Egyptians (around 1550 B.C.) and the ancient Greeks (around 413 B.C.). These early descriptions noted the association between fevers and wet ground. In fact, the word "malaria" actually derives from the Italian for "bad air"-- the mal'aria associated with marshes and swamps.

A single-celled parasite known as a sporozoan causes malaria. This sporozoan belongs to the genus Plasmodium, and the four species that threaten humans are P. falciparum, P. malariae, P. vivax and P. ovale. Of these four, P. falciparum and P. vivax are the most common, and P. falciparum is by far the most dangerous.

Mosquitoes alone spread malaria in nature. (The disease can be transmitted unnaturally through shared needles or by blood transfusion from infected donors.) When a mosquito bites an infected individual, the sporozoan's male and female sexual stages, or gametocytes, are taken up in the blood meal. Fertilization ensues in the mosquito's gut, and an "ookinete" forms. The ookinete then bores through the mosquito's stomach wall, becoming an oocyst, which subsequently divides to produce about a thousand infective sporozoites. In P. falciparum this process takes five to seven days, after which the sporozoites are released. They then migrate to the insect's salivary glands. Because mosquitoes inject their saliva when they bite (it contains anticoagulants and local anesthetic substances that facilitate blood sucking), the malaria sporozoites will be passed along to the mosquito's next victim.

Once inside the bloodstream of the bitten individual, the sporozoites home in on the liver. Each sporozoite invades a separate liver cell, and in P. falciparum takes five to seven days to divide and produce thousands of "merozoites," each of which will infect a red blood cell (erythrocyte) when the liver cell bursts. After entering the erythrocyte, the merozoite breaks down the cell's hemoglobin, feeding off the amino acids. The growing parasite, or trophozoite, will eventually become a "shizont" when it begins to divide again to form new merozoites. This erythrocytic cycle takes a variable amount of time in different malaria species--48 hours in P. falciparum but 72 hours in P. malariae infections. (One rare, sneaky exception to this progression can occur in P. vivax or P. ovale infections: when the sporozoite invades the liver cell, it does not produce merozoites immediately but may linger for a year or more in the liver before activating. This stage is known as a hypnozoite and can cause a relapse of malaria many months after an apparent cure.)

Characteristic signs of malaria infection are fever and flu-like symptoms, including headaches and muscle or joint pain. These usually begin after an incubation period of 10 to 14 days after the infective bite, during which the malaria parasite first inhabits the liver and then quietly multiplies in the blood. Classically, the fever is intermittent, recurring every few days, corresponding to the erythrocytic cycle. Each time the infected cells burst, liberating new merozoites, toxic metabolites and malarial antigens are also released. The body's immune system responds with a fever. In P. falciparum infections, fevers would occur on days 1, 3 and 5, whereas in P. malariae, fevers would occur on days 1, 4 and 7, and so forth. It is important, however, to remember two points. First, malarial fevers, especially in P. falciparum infections, do not always show cyclic temperature changes. Second, P. falciparum malaria can kill within 48 hours of the first signs, so it is essential that medical help is sought if one develops such symptoms after a visit to a region where malaria is a problem.

Alphonse Laveran, a French army doctor, described the malarial parasite--and proposed that it caused malaria--in 1880. But the final piece of the puzzle was put into place by a British physician, Sir Ronald Ross, who was working in India in 1897 when he observed the development of oocysts in mosquitoes that had been fed on infected individuals. Ross's description of the complete life cycle of the malarial parasite won him the Nobel Prize for Medicine in 1902.


Malaria Control During Mass Population Movements and Natural Disasters (2003)

A familiarity with the technical aspects of malaria is necessary for decisions to be made about how to devise a locally appropriate malaria control strategy. Such familiarity can aid in determining whether malaria might pose an important public health risk in a given situation, how large a problem it might be, which drugs would be expected to be effective and which probably would not, and which groups within a displaced population might be at increased risk of malaria and therefore need special attention.

The following section presents information on areas at risk of malaria transmission, the mechanics of how malaria is transmitted within a population, the range of clinical manifestations of malaria, antimalarial drug resistance, and the mosquito vector. The information presented is by no means exhaustive but should aid in basic decision making or in recognizing when expert assistance is needed.

AREAS AT RISK

Malaria occurs primarily in tropical and some subtropical regions of Africa, Central and South America, Asia, and Oceania (Figure 2-1). In areas where malaria occurs, there is tremendous variation in the intensity of transmission and risk of infection. For example, over 90 percent of clinical malaria infections and deaths occur in sub-Saharan Africa (World Health Organization, 1996a). However, even there the risk varies widely. Highland (>1,500 m) and arid areas (<1,000 mm rainfall/year) typically have

less malaria, although these areas are prone to epidemic malaria if climactic conditions become favorable to mosquito development (World Health Organization, 1996a). Although urban areas have typically been at lower risk, explosive unplanned population growth has been a major factor in making urban or peri-urban transmission an increasing problem (Knudsen and Sloof, 1992).

Human malaria is caused by one or more of four parasites: Plasmodium falciparum, P. vivax, P. ovale, and P. malariae. Distribution of these parasites varies geographically, and not all species of malaria are transmitted in all malarious areas. P. falciparum, the species most commonly associated with fatal malaria, is transmitted at some level in nearly all areas where malaria occurs. It accounts for over 90 percent of all malaria infections in sub-Saharan Africa, for nearly 100 percent of infections in Haiti, and causes two-thirds or more of the malaria cases in Southeast Asia. P. vivax is a relatively uncommon infection in sub-Saharan Africa. Duffy antigens, which are required by the parasite to invade red blood cells, are lacking in many ethnic groups, especially in West Africa. Vivax malaria, however, is the predominant species in Central America, most of malarious South America, and the Indian subcontinent (Miller et al., 1977).

MECHANISMS OF INFECTION AND TRANSMISSION

Malaria is typically transmitted by the bite of an infective female Anopheles mosquito transmission can also occur transplacentally, as a result of blood transfusion, or by needle sharing. Infective mosquitoes inject sporozoites into the bloodstream during feeding (see Figure 3-1). These sporozoites infect liver cells (b) where they undergo asexual reproduction (exoerythrocytic schizogony), producing schizonts (c). In 6 to 14 days (sometimes longer), the schizonts rupture, releasing merozoites into the bloodstream (d). Merozoites invade red blood cells and undergo a second phase of asexual reproduction (erythrocytic schizogony), developing into rings (e), trophozoites (f), and finally blood stage schizonts (g). The schizonts rupture, destroying the red blood cell and releasing more merozoites into the bloodstream, starting another cycle of asexual development and multiplication (h). This erythocytic cycle will continue until the infected individual is successfully treated, mounts an immune response that clears the infection, or dies. During this cycle, sexual forms called gametocytes are produced (i) and can be ingested by a mosquito during a

FIGURE 3-1 Malaria Life Cycle.

blood meal (j). Sexual reproduction occurs in the mosquito (k). Sporozoites are formed (l), which migrate to the salivary glands, making the mosquito infective to humans.

The timing of events in the life cycle of malaria parasites and the number of merozoites produced during schizogony vary by species. Additionally, two species of malaria, P. vivax and P. ovale, have a form, &ldquohypnozoites&rdquo (m), that can persist in the liver for months to years, causing periodic relapses of peripheral parasitemia and illness (see Table 3-1).

MALARIA VECTORS AND VECTOR BEHAVIOR

Human malaria is transmitted by the bite of female mosquitoes belonging to the genus Anopheles. Of the 400 or so species of Anopheles in the world, approximately 60 are important vectors of malaria. However, a particular species of Anopheles may be an important vector in one area of the world and of little or no consequence in another.

Different species of Anopheles can behave differently. Mosquito behavior can differ in terms of breeding or larval habitat (e.g., fresh vs. brackish water flowing streams, still pools, or man-made habitats shaded or sunny sites), feeding preferences (e.g., time of day when peak biting occurs, preferences for people over animals, feeding indoors or outside), and resting habits (resting indoors after feeding or leaving the house before resting). These differences in mosquito behavior can affect both the epidemiology of malaria and the choice of malaria control strategy used. For example, An. dirus is an important vector in Southeast Asia and is primarily a forest dweller. People at greatest risk are, therefore, those who enter the forest for whatever reason, while those who stay closer to home (such as small children) are at less risk. This also means that malaria control strategies aimed at preventing mosquito biting in the home (such as residual spraying or insecticide-treated bed nets) would be of little value in preventing exposure and infection. An. gambiae, the most important vector in much of sub-Saharan Africa, breed in small temporary pools of water (even as small and temporary as cattle hoofprints). Therefore, vector control strategies aimed at reducing or eliminating breeding sites will likely have little impact. For these reasons, expert advice relevant to the primary malaria vectors in a given area is essential for making sound decisions regarding control options.

TABLE 3-1 Characteristics of the Four Species of Human Malaria

12-17 (15) Days to 6-12 months

Severity of primary attack

Duration of primary attack a

Duration of untreated infection a

Central nervous system complications a

a Influenced by immunity. Documentation of complications for species other than P. falciparum is limited.

SOURCE: Adapted from Bruce-Chwatt (1985).

VECTOR LIFE CYCLE

There are four stages in the life cycle of the mosquito: egg, larva, pupa, and adult. Eggs are deposited singly on water in suitable breeding sites, where the developing embryo hatches as a larva after 2 or more days. During the aquatic period of development, the larva sheds its skin four times. The fourth larval molt gives rise to a pupa. At this stage the mosquito undergoes a complete metamorphosis, emerging as an adult. When parasites are ingested during a blood meal, they undergo further development in the mosquito&rsquos stomach, and during the next 10 to 20 days the parasite passes through a number of stages, eventually multiplying and penetrating all parts of the mosquito body. Parasites that end up in the salivary glands can be transmitted to humans when the mosquito takes another blood meal.

The length of each developmental stage depends on temperature and humidity. The life span of adults under natural conditions is difficult to determine but averages 10 to 14 days or longer (Service, 1993). The time for an egg to develop to an adult in the tropics can be as short as 5 to 7 days. Development of malaria parasites in the mosquito host is also temperature dependent as ambient temperatures decrease, the time needed for parasites to develop increases. Malaria transmission stops when the time needed for development of infective sporozoites exceeds the life span of the mosquito (Gilles, 1993).

MALARIA ILLNESS

The normal incubation period from infective mosquito bite to onset of clinical symptoms is 9 to 30 days or longer, depending on such parameters as species of parasite, immune status, infecting dose, and use of antimalarial drugs. The clinical symptoms associated with malaria parasites are produced by increases in cytokines (particularly tumor necrosis factor) in response to merozoites, pyrogens, and cellular debris released when red blood cells rupture at the end of schizogony (Kwiatkowski, 1990). Onset of illness is associated with the initial rupture of erythrocytic schizonts exoerythrocytic forms (sporozoites, exoerythrocytic schizonts, and hypnozoites) and gametocytes do not cause clinical symptoms.

Typical symptoms among nonimmune individuals include fever, chills, myalgias and arthralgias, headache, diarrhea, and other nonspecific signs. Other findings may include splenomegaly, anemia, thrombocytopenia,

pulmonary or renal dysfunction, changes in mental status, and coma. Most cases of malaria-related severe illness and death are associated with P. falciparum infection. This is due, in part, to the parasite&rsquos ability to infect both mature and immature red blood cells, its rapid rate of asexual reproduction, and a broad range of poorly understood pathological processes associated with its ability to sequester in postcapillary venules, especially in the central nervous system. Cyclical fevers seen in nonimmune individuals with synchronous infections (occurring when a majority of schizonts rupture at the same time) are frequently absent among individuals with immunity.

In general, immunity to malaria is acquired after repeated exposure to the malaria parasite those individuals who survive their initial infections develop some degree of immunity. In highly endemic areas, most clinical malaria and malaria-associated mortality occurs in children less than 5 years old, whose immunity has not yet fully developed. Although malaria prevalence in endemic areas decreases with increasing age, suggesting an acquired ability to clear malaria infections, immunity to malaria typically also involves the development of a tolerance for the presence of malaria parasites in the blood with a minimum of symptoms and relative protection from severe illness and death.

EPIDEMIOLOGY OF CLINICAL MALARIA

Malaria transmission intensity, levels of acquired immunity in a population, and manifestations of malaria illness are intimately linked (see Table 3-2 Snow et al., 1994 Slutsker et al., 1994). Understanding this relationship should help in estimating the likely impact of malaria in a given population. An important additional consideration is understanding the implications of differences between the environment from which a displaced population comes and the environment in which that population settles, even if only temporarily.

The overall level of immunity to malaria is highest in areas where malaria transmission is the most intense. The first exposure to malaria occurs very early in childhood, and with repeated exposures the likelihood of severe illness or death lessens. In Africa, where the majority of malaria-associated deaths occur, the highest mortality rates occur in children less than 5 years old. It has been estimated that in the Gambia, malaria accounts for 25 percent of all deaths among children less than 5 years old. Malaria-associated mortality decreases rapidly with increasing age (Greenwood et

TABLE 3-2 Malaria Transmission Characteristics, Immunity, and Clinical Features

Areas Where P. falciparum Malaria Is Predominant or Represents a Significant Proportion of Infecting Malaria Species

Areas Where Nonfalciparum Malaria Is the Predominant Infecting Species

Areas of Intense Perennial Malaria Transmission and/or Populations with High Levels of Immunity Originating in Those Areas

Areas of Low-to-Moderate Malaria Transmission

Areas of Very Low, Highly Seasonal, or Epidemic Malaria Transmission and/or Low Levels of Immunity in the Population

Examples: Central America, some parts of the Indian subcontinent, parts of Central Asia, parts of eastern Europe.

Typical epidemiology of clinical illness:

Nonfalciparum malaria can be a significant cause of morbidity but is rarely associated with severe illness or death. Spontaneous splenic rupture can occur in chronic P. vivax infections, but cerebral malaria has only rarely been attributed to this parasite. Infections caused by P. malariae have been associated with nephrotic syndrome. In areas where both falciparum and nonfalciparum malaria occur, species-specific treatment is advisable. Because drugs used for the treatment of acute infections do not typically affect the hypnozoites of P. ovale and P. vivax, these infections can relapse months to years later.

Example: Most of tropical equatorial Africa.

Typical epidemiology of clinical illness:

Bulk of clinical disease occurs in young (<5 years old) children. While adults and older children are often parasitemic, they are more likely to be asymptomatically infected or to have only mild symptoms. Severe anemia tends to be more common than cerebral malaria in young children.

Examples: Parts of southern Africa, coastal eastern Africa, Indian subcontinent, Southeast Asia.

Typical epidemiology of clinical illness:

Bulk of disease occurs in broader range of ages. Cerebral malaria becomes more common.

Examples: Sahelian and highland areas of Africa, Central America.

Typical epidemiology of clinical illness:

Disease can occur in all age groups and is more likely to progress to severe disease if not promptly treated. In some areas of very low transmission (e.g., Haiti), mortality rates associated with malaria can be paradoxically low, probably due to high incidence of clinical symptoms, rapid treatment-seeking behavior of the population, and the high efficacy and ready availability of CQ.

al., 1987 Campbell, 1991). In areas with very high levels of transmission, severe malaria tends to manifest itself more frequently as anemia than as cerebral disease. As transmission intensity decreases and population immunity is lessened, illness is seen more frequently in all age groups and the incidence of cerebral disease increases relative to severe anemia.

Displaced populations coming from areas of little or no malaria transmission to areas with intense transmission are therefore at greatest risk of severe illness and death due to a lack of acquired immunity. Until they themselves acquire a protective level of immunity to malaria, these populations will probably require a much more intensive intervention effort to achieve and maintain low rates of morbidity and mortality.

Malaria also has important effects in the way of chronic or repeated infections, including anemia (especially among individuals with underlying malnutrition), and poor pregnancy outcomes. Anemia, itself an important cause of mortality associated with malaria, is also a common reason for blood transfusion.

MALARIA DURING PREGNANCY

Among nonimmune women, acute malaria during pregnancy carries a high risk of maternal and fetal death if not treated promptly and adequately. Among semiimmune pregnant women, however, malaria parasites preferentially sequester in the placenta, with minimal increase in overt clinical disease. Malaria during pregnancy is a cause of both maternal anemia and low birth weight, accounting for as much as 35 percent of preventable low birth weight in malarious areas (Steketee et al., 1996a). Low birth weight, in turn, is a well-recognized risk factor for infant mortality (McCormick, 1985). In most populations living in areas of high malaria transmission, this is an issue primarily affecting women in their first and second pregnancies.

MALARIA AND HIV/AIDS

Malaria and HIV infection can occur at high frequencies in the same population, especially in sub-Saharan Africa, raising concerns that interactions between these two diseases could greatly complicate control of both (Corbett et al., 2002). Malaria and HIV have indeed been shown to interact in important ways. Peripheral malaria infection has been shown to occur more frequently and parasite densities have been shown to be higher

among HIV-positive pregnant women compared with HIV-negative pregnant women (Steketee et al., 1996b). HIV-infected women are at increased risk of placental malaria, even during later pregnancies when placental sequestration is less of a problem among HIV-negative women (Steketee et al., 1996b). Similarly, HIV-infected, nonpregnant individuals are at increased risk of both malaria infection and illness (Whitworth et al., 2000). There is also evidence that acute malaria infection can increase viral load among HIV-infected individuals, an increase that is reversed with effective malaria therapy (Hoffman et al., 1999). Finally, unscreened blood transfusions for malaria-associated anemia remain an important source of HIV transmission in malarious areas (Corbett, et al., 2002).

ANTIMALARIAL DRUG RESISTANCE

Simply defined, antimalarial drug resistance occurs when malaria parasites gain the ability to survive what should be an effective dose of antimalarial drugs. Resistance occurs because of naturally occurring mutations that affect the susceptibility of the parasite to a given drug. For some drugs, resistance can occur after a single-point mutation (as for the drug atovaquone) with others a number of mutations may be required. Factors that facilitate intensification of drug resistance include poor adherence to recommended treatment regimens, inadequate dosing, use of poor-quality drugs, presumptive treatment, and use of drugs that have a long half-life. Because of rapidly developing and spreading resistance to antimalarials and the relatively slow process of developing new antimalarials, the number of useful drugs is dwindling (Bloland and Ettling, 1999 Winstanley, 2000). The most commonly available antimalarial drugs are described in Appendix A and Table 3-3, although not all are practical or appropriate for use in any given situation.

Chloroquine-resistant P. falciparum (CRPF) was first recognized almost simultaneously in Thailand and South America in the late 1950s. It was first identified on the east coast of Africa in 1978. In the past 20 to 25 years, CRPF has spread and intensified to the point that only Central America northwest of the Panama Canal, the island of Hispaniola (Haiti and the Dominican Republic), and limited regions of the Middle East are free of chloroquine (CQ) resistance. All other endemic areas have malaria that is, to varying extents, resistant to CQ. In some regions, CQ resistance has intensified to the point where the drug no longer has an optimal effect

TABLE 3-3 Antimalarial Drugs for Uncomplicated Malaria

Combination Therapy

Treatment of nonsevere falciparum infections thought to be CQ and SP-resistant.

15 mg/kg MQ base on day 2 of treatment, followed by 10 mg/kg MQ base on day 3. Usual adult dose is 750 mg on day 2 followed by 500 mg on day 3.

15 mg/kg MQ on day 2 of treatment followed by 10 mg/kg MQ on day 3.

Safety of artemisinins and MQ during first trimester of pregnancy not established. Vomiting after MQ can be reduced by administering it on the second and third days after an initial dose of artesunate.

Artemisinin (20 mg/kg initially followed by 10 mg / kg once daily for 2 more days) can be substituted can besubstituted for artesunate.

4 mg/kg artesunate daily for 3 days.

4 mg/kg artesunate daily for 3 days.

Treatment of nonsevere falicparum infections thought to be CQ resistant.

As for SP monotherapy (see below).

As for SP monotherapy (see below).

This combination has not been evaluated as extensively as MQ + artesunate.

Safety of artemisinin during first trimester of pregnancy not established.

Artemisinin (as described above) can be substituted for artesunate.

4 mg/kg artesunate daily for 3 days.

4 mg/kg artesunate daily for 3 days.

CQ or amodiaquine + artesunate

Treatment of CQ-resistant malaria.

Treatment: 25 mg base/kg divided over 3 days.

Treatment: 25 mg base/kg divided over 3 days.

Combination of amodiaquine + artesunate generally more useful due to widespread, typically high-level CQ resistance. In some areas,

Average adult dose: 2.5 gm (salt) divided over 3 days.

neither would be expected to be particularly effective.

See note below on possible toxicity associated with amodiaquine.

4 mg/kg artesunate daily for 3 days.

4 mg/kg artesunate daily for 3 days.

Lumefantrine/ artemether also known as: co-artemether

Trade names: Coartem, Riamet

Treatment of multidrug-resistant malaria.

Adult: 4 tablets per dose at 0, 8, 24, 36, 48, and 60 hours.

Pediatric: 10-14 kg: 1 tablet/dose 15-24 kg: 2 tablets/dose 25-35 kg: 3 tablets/dose >35 kg: as for adult given at 0, 8, 24, 36, 48, and 60 hours.

Hyper-sensitivity to component drugs.

Commercially available fixed-dose combination of 20 mg artemether and 120 mg lumefantrine.

Not recommended for <10 kg or pregnant women

Available in 2 packaging schemes: 24 tablet/6 doses (Riamet) and 16 tablets/

4 doses. (Coartem)&mdash 4 dose regimen not as effective especially for nonimmunes.

Also available from WHO in specially designed blister packs.

CQ (or amodiaquine) + sulfadoxine/ pyrimethamine

Treatment of CQ-resistant malaria.

Use routine doses for both CQ/ amodiaquine and SP.

Use routine doses for both CQ/ amodiaquine and SP.

Same as for CQ/ amodiaquine and SP monotherapy.

Primarily useful only in areas where CQ resistance is low to moderate and SP resistance is low.

See notes of caution regarding use of amodiaquine.

Single-Agent Therapy

CQ Trade names: Nivaquine, Malaraquine, Aralen, many others

Treatment of nonfalciparum infections. Treatment of P. falciparum infections in areas where CQ remains effective.

Treatment: 25 mg base/kg divided over 3 days.

Average adult: 1.5 gm base divided over

Treatment: 25 mg base/kg divided over 3 days.

Widespread resistance in P. falciparum in most regions. Resistance in P. vivax occurs in some areas.

Can cause pruritus in dark-skinned patients, reducing compliance.

Chemoprophylaxis in areas where CQ remains effective.

Preparations differ in amount of base (100 or 150 mg tablets, 50 mg syrup).

Prophylaxis: 5 mg/kg base per week.

Prophylaxis: 5 mg base/kg once per week.

Amodiaquine Trade names: Camoquine, others

Treatment of nonsevere falicparum infections thought to be CQ resistant.

Treatment: 25 mg base/kg divided over 3 days.

Single dose of 25 mg (sulfa)/kg

Treatment: 25 mg base/kg divided over 3 days.

Single dose of 25 mg (sulfa)/kg

Cross-resistance with CQ limits usefulness in areas with high rates of CQ resistance. Has been associated with toxic hepatitis and agranulocytosis when used as prophylaxis risk when used for treatment unknown.

SP Trade name: Fansidar, others

Treatment of nonsevere falicparum infections thought to be CQ resistant.

Average adult: 3 tablets as a single dose.

Efficacy for vivax infections may be poor.

Widespread resistance in P. falciparum in some regions.

Sulfalene/ pyrimethamine (Metakelfin)

Can cause severe skin disease when used prophylactically risk when used as treatment unknown but likely to be very low.

MQ Trade names: Larium, Mephaquine

Treatment of nonsevere falicparum infections thought to be CQ and SP resistant.

Chemoprophylaxis in areas with CQ resistance.

Treatment: 750 to 1,500 mg base depending on local resistance patterns. Larger doses (>15 mg/kg) best given in split doses over 2 days.

Treatment: 15-25 mg base/kg depending on local resistance patterns. Larger doses (>15 mg/kg) best given in split doses over 2 days.

Known or suspected history of neuro-psychiatric disorder, history of seizures, concomitant use of halofantrine.

Vomiting can be a common problem in young children. In some populations (e.g., very young African children), unpredictable blood levels, even after appropriate dosing, can produce frequent treatment failure.

Prophylaxis: 250 mg once per week.

Prophylaxis: 5 mg base/kg once per week.

Halofantrine Trade name: Halfan

Treatment of suspected multidrug-resistant falciparum.

8 mg base/kg every 6 hours for 3 doses.

Average adult: 1,500 mg base divided into 3 doses as above.

8 mg base/kg every 6 hours for 3 doses.

Preexisting cardiac disease, congenital prolongation of QTc interval, treatment with MQ within prior 3 weeks, pregnancy.

Cross-resistance with MQ has been reported. Reported to have highly variable bioavailability. Risk of fatal cardiotoxicity.

Treatment of severe malaria. Treatment of multidrug-resistant P. falciparum. Treatment of malaria

Nonsevere malaria: 8 mg base/kg 3 times daily for 7 days.

Nonsevere malaria: 8 mg base/kg 3 times daily for 7 days.

Side effects can greatly reduce compliance.

Used in combination with tetracycline, doxycycline, clindamycin, or SP (where effective) and in areas where

during first trimester of pregnancy.

Average adult: 650 mg 3 times daily for 7 days.

Severe: see section on treatment of severe malaria.

quinine resistance is not prevalent, duration of quinine dosage can be reduced to 3 days.

Severe: see section on treatment of severe malaria.

Tetracycline (tetra)/ doxycycline (doxy)

In combination with quinine, can increase efficacy of treatment in areas with quinine resistance and/or reduce likelihood of quinine-associated side effects by reducing duration of quinine treatment. Prophylaxis.

Tetra: 250 mg/ 4 times per day for 7 days.

Tetra: 5 mg/kg 4 times per day for 7 days.

Age less than 8 years, pregnancy.

Used only in combination with a rapidly acting schizonticide such as quinine.

Doxy: 100 mg 2 times per day for 7 days.

Doxy: 2 mg/kg twice per day for 7 days.

Prophylaxis: 100 mg doxy per day.

Prophylaxis: 2mg/kg doxy per day up to 100 mg.

For patients unable to take tetracycline. In combination with quinine, can increase efficacy of treatment in areas with quinine resistance and /or reduce likelihood of quinin-associated side effects by reducing duration of quinine treatment.

300 mg 4 times per day for 5 days.

20 to 40 mg/kg per day divided into 3 daily doses for 5 days.

Severe hepatic or renal impairment, history of gastrointestinal disease, especially colitis.

Not as effective as tetracycline, especially in nonimmune patients. Used only in combination with a rapidly acting schizonticide such as quinine.

Atovaquone/ proguanil Trade name: Malarone

Treatment of multidrug-resistant P. falciparum infections.

4 tablets (1,000 mg atovaquone + 400 mg proguanil) daily for 3 days.

No pediatric formulation currently available, but for patients 11-40 kg in body weight:

Fixed-dose combination. Reportedly safe in pregnancy and young children.

Drug donation program exists.

Pediatric formulation in development.

11-20 kg 1/4 adult dose 21-30 kg 1/2 adult dose 31-40 kg 3/4 adult dose

Treatment of multidrug-resistant P. falciparum infections.

4 mg/kg on day 1 followed by 2 mg/kg daily for total of 7 days.

4 mg/kg on day 1 followed by 2 mg/kg daily for total of 7 days.

A high number of counterfeit artemisinin products have been found in Southeast Asia.

20 mg/kg on day 1 followed by 10 mg/kg daily for total of 7 days.

20 mg/kg on day 1 followed by 10 mg/kg daily for total of 7 days.

Use of artemisinin derivatives not recommended during first trimester of pregnancy.

4 mg/kg on day 1 followed by 2 mg/ kg daily for total of 7 days.

4 mg/kg on day 1 followed by 2 mg/kg daily for total of 7 days.

Rectal preparations of artemisinin and artesunate are available for initial treatment of severe malaria in facilities unable to administer parenteral therapy.

4 mg/kg on day 1 followed by 2 mg/kg daily for total of 5 to 7 days.

4 mg/kg on day 1 followed by 2 mg/kg daily for total of 5-7 days.

Treatment of P. vivax infections (reduce likelihood of relapse). Gametocytocidal agent.

14 mg base per day for 14 days.

45 mg once per week for 8 weeks.

0.3 mg (base)/kg daily for 14 days.

G6PD deficiency, pregnancy.

Primaquine has also been investigated for prophylaxis use. See text for additional cautions.

b Cost is given for a full adult (60-kg) treatment course. Prices derived from individual reports, personal communications, McFayden (1999), Medical Economics Co. (1999) and WHO (2001b). Prices reflect best prices or best estimates local prices may differ greatly.

on P. falciparum malaria parasites and can no longer be relied on to provide effective treatment or prophylaxis.

Drug resistance is not limited to chloroquine. In some areas of Southeast Asia, the situation is deteriorating to the point where few effective therapies exist. Chloroquine was abandoned as first-line therapy for malaria in Thailand in 1972 in preference to sulfadoxine/pyrimethamine (SP). Drug resistance develops rapidly to dihydrofolate reductase inhibitors (such as pyrimethamine and proguanil) when used alone or in combination with sulfa drugs (such as SP) (Björkman and Phillips-Howard, 1990 Sibley et al., 2001). In 1985, in response to declining SP efficacy, SP was combined with mefloquine (Thaithong et al., 1988). After a few years of widespread use of mefloquine (15 mg/kg), greater than 50 percent of P. falciparum infections showed resistance to it in some areas of Thailand (Mockenhaupt, 1995). Cure rates were improved to 70 to 80 percent by increasing the dose of mefloquine to 25 mg/kg, but the efficacy of this higher dose also declined rapidly in some areas (Nosten et al., 2000). In Southeast Asia, parasitological response to quinine has also been deteriorating (Bunnag and Harinasuta, 1987 Wongsrichanalai, et al., 2002). Resistance to newer antimalarials, such as halofantrine, has also been reported, especially in areas with mefloquine resistance (Wongsrichanalai et al., 1992 ter Kuile et al., 1993).

Currently, multidrug-resistant malaria in Thailand is being treated with a combination of an artemisinin derivative and mefloquine efficacy of this combination has remained high (Nosten et al., 2000). To date, no confirmed cases of resistance to the artemisinin drugs have been reported. However, reports of decreasing sensitivity in vitro in some areas, as well as a number of case reports of potential (but unconfirmed) treatment failures, raise concern that this class of antimalarial drug is not &ldquoimmune&rdquo to the development of resistance (Wongsrichanalai et al., 1999 Sahr et al., 2001).

In many areas where population displacement occurs, national treatment policies of the host and source countries may not necessarily reflect the drug resistance patterns of a given region or population. In many cases this is due to a lack of current efficacy data, a lack of funds for implementation of a new policy, and a variety of other concerns (Bloland and Ettling, 1999). For example, CQ is still the recommended first-line treatment for P. falciparum in much of Africa, despite the high prevalence of CRPF. Choice of the most appropriate antimalarial drug should be based, whenever possible, on actual evaluation of the efficacy of possible therapeutic options using standard methods (see Appendix B).

Drug resistance is not an all-or-nothing phenomenon. In any given area, a wide range of parasitological responses can be found, from complete sensitivity to high-level resistance (see Table 3-4). In general, malaria parasites in western sub-Saharan Africa are less resistant to drugs like CQ and SP than malaria parasites in eastern or southern Africa. In Southeast Asia the distribution of drug resistance is highly variable and focal. While some very well-publicized areas (such as the refugee camps along the Thai-Burmese border) are faced with highly resistant malaria, requiring complicated combination therapy approaches, nearby areas reportedly have had a much longer period of success with MQ alone (K. Thimasarn, Malaria Control Programme, Thai Ministry of Public Health, 1998, personal communication Singhasivanon, 1999 Wongsrichanalai et al., 2000).

The policy response to increasing evidence of antimalarial drug resistance has been variable as well. In parts of East Africa, parasitological resistance to CQ is very high, with 80 to 90 percent of P. falciparum infections being moderately to highly resistant (Bloland et al., 1993). In response to these high rates of resistance, Malawi switched from CQ to SP for first-line therapy for P. falciparum in 1993. A number of countries in eastern and southern Africa (including Tanzania, Kenya, Democratic Republic of Congo, Rwanda, Uganda, Ethiopia) have made similar policy changes to SP alone or in combination (with either CQ or amodiaquine) on a national or provincial/district level. After a long period of disinclination to change treatment policies, many more countries in sub-Saharan Africa are now reevaluating their national treatment guidelines and considering policy changes to locally effective regimens. Although the drugs being used differ, similar efforts are under way in the Amazon region and Southeast Asia.

VECTOR CONTROL

Attempts to interfere with the entomological link in malaria transmission are an essential and integral part of many malaria control programs. However, a vector control measure that is appropriate in one setting may be totally inappropriate elsewhere (see earlier section on Malaria Vectors and Vector Behavior). It is therefore strongly advised that the assistance of an experienced medical entomologist who is familiar with existing malaria vector data for the area in question be obtained. Decisions based on expert advice and firsthand data are the most certain route to cost-effective vector control.

The importance of understanding vector biology and behavior prior to initiating control measures is evident when one considers the key questions that must be answered to determine which type of control measure is best for a given situation. Identification of the mosquito species responsible for most malaria transmission involves surveillance, collection, and species identification from various parts of the affected area. There can also be a number of anophelines in a given area. However, not all anopheline mosquitoes transmit malaria and not all anophelines that do transmit malaria are efficient vectors. Therefore, the most commonly collected adult anopheline species captured in a given area will sometimes, but not always, be responsible for most malaria transmission. Control activities aimed at one important vector species may not be effective against another vector species. For example, insecticide-treated nets may be useful for reducing exposure to mosquitoes biting inside houses but may have no effect on mosquitoes biting primarily outside.

Information about the breeding places of vectors is required. Mosquitoes have diverse breeding habits, some of which may be targeted for control purposes. Mosquitoes generally do not range farther than 2 to 3 km from their breeding sites, unless carried by winds or some other vehicle. When one site or a few produce most of the vectors responsible for transmission, larval control may be appropriate and effective. In situations where breeding sites are small, dispersed, or not easily identified, however, larval control would be inappropriate. As mentioned previously, some malaria vectors (such as An. gambiae in Africa) can breed in pools as small and temporary as an animal&rsquos hoofprint. Identifying breeding areas also requires considerable skill. When done correctly, larval control has been very effective in certain circumstances, such as special projects, urban areas, and land-based business ventures.

Differences in the behavior patterns of adult mosquitoes have a marked effect on their capacity to transmit malaria as well as the choice of control methods used. Preferred time of biting, for example, can vary from daytime to late evening. The efficacy of many control measures varies, depending on the mosquito activity cycle. Insecticide-treated bed nets or other control measures that are used indoors, such as space spraying with insecticides, would obviously have little effect on malaria transmission occurring at times when people are not indoors. For example, malaria transmission in much of Southeast Asia is associated with exposure to mosquitoes during activities conducted in the forest.