Newer approaches to malaria control.

Malaria is the third leading cause of death due to infectious diseases affecting around 243 million people, causing 863,000 deaths each year, and is a major public health problem. Most of the malarial deaths occur in children below 5 years and is a major contributor of under-five mortality. As a result of environmental and climatic changes, there is a change in vector population and distribution, leading to resurgence of malaria at numerous foci. Resistance to antimalarials is a major challenge to malaria control and there are new drug developments, new approaches to treatment strategies, combination therapy to overcome resistance and progress in vaccine development. Now, artemisinin-based combination therapy is the first-line therapy as the malarial parasite has developed resistance to other antimalarials. Reports of artemisinin resistance are appearing and identification of new drug targets gains utmost importance. As there is a shift from malaria control to malaria eradication, more research is focused on malaria vaccine development. A malaria vaccine, RTS,S, is in phase III of development and may become the first successful one. Due to resistance to insecticides and lack of environmental sanitation, the conventional methods of vector control are turning out to be futile. To overcome this, novel strategies like sterile insect technique and transgenic mosquitoes are pursued for effective vector control. As a result of the global organizations stepping up their efforts with continued research, eradication of malaria can turn out to be a reality.

INTRODUCTION

Malaria is undoubtedly a scourge of mankind from prehistoric times. Evolutionary biologists estimate that Plasmodium was causing human disease for the last 100,000 years or so and Plasmodium falciparum strains causing lethal disease evolved some 4000 years ago.[1] This corresponds to the natural selection of G6PD deficient alleles in African population between 4000 and 12,000 years ago. The oldest genomic evidence of malaria was obtained from the mummy of King Tutankhamun, an Egyptian pharaoh who died around 1324 BC.[2] Malaria has had a profound impact on the recent evolution of mankind, affecting the distribution of certain conditions like G6PD deficiency, thalassemias, sickle cell trait, hemoglobin C, etc.

THE PROBLEM

Once the leading cause of death due to infectious agents, malaria is now ranked the third leading cause of death due to an infectious agent after HIV and tuberculosis. World Health Organization (WHO) estimates the global incidence of malaria as 243 million cases with a whopping 863,000 deaths worldwide in 2008.[3] High incidence is seen in the African countries with 89% of deaths occurring in them. An estimated 85% of malarial deaths occur in children below 5 years of age and is an important contributor to the under-five mortality.[4] WHO places 3.3 billion people, i.e., 50% of the world population at risk of contracting malaria, with 2.1 billion at a low risk and 1.2 billion at a high risk. During 2009, India had reported 1.56 million confirmed cases with 1144 deaths,[5] but WHO estimates that only 10-12% of cases are reported in India.[3] A recent paper has estimated that the malarial deaths in India could be as high as 205,000 per year.[6] This estimation was based on field visits by the investigators rather than the deaths reported in hospitals, as was estimated by WHO. If this study is to be believed, then the number of malarial deaths worldwide has to be revised to a much higher value based on data from field visits. Due to environmental and climatic changes, there is also a change in vector population and distribution leading to resurgence of malaria in numerous foci.[7] The number of malaria cases imported into developed countries is also on the rise due to increased international travel.[7]

The global fight against malaria is lead by various organizations like the WHO and the Roll Back Malaria Partnership. In India, the National Vector Borne Diseases Control Programme is entrusted with malaria control. To control malaria in high incidence states, the World Bank funded Enhanced Malaria Control Project was launched in 1997. Though the program is successful in a few states, it is not successful in some states like Orissa, Jharkhand and Chhatisgarh due to shortcomings in the project implementation.[8]

This article discusses about the global action plan for malaria eradication, newer approaches including the development of new drugs, the combination therapy to combat resistance (especially that of artemisinin), newer drug targets, vaccine development and various measures for vector control.

PLASMODIUM

Although there are more than 200 species of Plasmodium, natural human disease is caused only by four species - P. falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale. Or so it was thought until recently when a species called Plasmodium knowlesi was found to cause natural human infection in Malaysian Borneo region. P. knowlesi is a malarial parasite of the Old World monkeys and accidental human infections have been reported earlier. Through nested polymerase chain reactions (PCRs), 28% of severe malaria infections and 85% of hyperparasitemic P. malariae infections were found to be P. knowlesi. These cases were misdiagnosed as P. malariae infections by routine microscopy. It causes high mortality due to hemolysis and hepatorenal dysfunction.[9] P. knowlesi is now identified as the fifth human malarial parasite.[10]

CURRENT TREATMENT GUIDELINES

Uncomplicated Falciparum malaria

The WHO has brought out guidelines for the treatment of malaria. The guidelines recommend the use of artemisinin-based combination therapy (ACT) for 3 days as first-line drugs against uncomplicated falciparum malaria.[11] The recommended ACTs are as follows:

artemether + lumefantrine

artesunate + amodiaquine

artesunate + mefloquine

artesunate + sulfadoxine-pyrimethamine

dihydroartemisinin + piperaquine

The recommended second-line treatments are

artesunate plus tetracycline or doxycycline or clindamycin – for 7 days and

quinine plus tetracycline or doxycycline or clindamycin – for 7 days.

In pregnancy, the recommended treatment is as follows:

First trimester:

Quinine plus clindamycin for 7 days

An ACT is indicated only if this is the only treatment immediately available or if treatment with 7-day quinine plus clindamycin fails or there is an uncertainty of compliance with a 7-day treatment.

Second and third trimesters:

ACTs or artesunate plus clindamycin or quinine plus clindamycin to be given for 7 days.

Severe Falciparum malaria

Artesunate 2.4 mg/kg IV or IM given on admission, 12 h and 24 h, then once a day

(or) Quinine 20 mg salt/kg on admission (IV or IM), then 10 mg/kg every 8 h

Parenteral antimalarials for at least 24 h followed by complete course of ACTs or oral quinine with doxycycline for 7 days

P. vivax malaria

Chloroquine sensitive

Whenever sensitive to chloroquine, Tab. chloroquine (150 mg) is preferred and the dose is 25 mg/kg divided over 3 days, combined with primaquine 0.25 mg/kg once daily for 14 days.

Chloroquine resistant

If P. vivax is resistant to chloroquine, ACT combined with primaquine is used.

Chemoprophylaxis

A combination of atovaquone and proguanil is recommended for the prophylaxis and treatment of falciparum malaria. Atovaquone 250 mg plus proguanil 100 mg is taken once daily starting from 2 days before travel, during and is continued for 7 days after returning from the endemic area.

Indian guidelines

The National Institute of Malaria Research, New Delhi, has brought guidelines for the diagnosis and treatment of malaria.[12] It differs from WHO guidelines in recommending chloroquine as the first-line therapy in sensitive areas.

Uncomplicated falciparum malaria

Chloroquine 25 mg/kg divided over 3 days + Primaquine 0.75 mg/kg on day 1

In resistant areas, the treatment consists of artesunate 4 mg/kg for 3 days + sulfadoxine (25 mg/kg) and pyrimethamine (1.25 mg/kg) on day 1 + primaquine 0.75 mg/kg on day 2.

The National Drug Policy on Malaria (2010)[13] recommends artesunate with sulfadoxine and pyrimethamine combination as the first-line of therapy in malaria.

Chemoprophylaxis

For chemoprophylaxis less than 6 weeks, the recommended regimen consists of doxycycline 100 mg daily, started 2 days before travel and continued for 4 weeks after leaving the endemic areas.

If it is for more than 6 weeks, the recommended regimen is mefloquine 250 mg weekly, started 2 weeks before and continued for 4 weeks after leaving the endemic areas.

Resistance to antimalarials

The plasmodia developed resistance to most of the antimalarials due to the widespread indiscriminate use of these drugs. Earlier, these drugs are used as monotherapy and this also contributed to the development of resistance. Chloroquine resistance appeared in the 1950s along the Thai-Cambodian border and Colombia and spread to the rest of the world.[14] The development of resistance to chloroquine in India was first reported from Assam in 1973.[15] By 1988, chloroquine resistance had spread to all of sub-Saharan Africa, and today, sensitive strains are present only in few regions. Resistance to chloroquine is mainly due to mutations in PfCRT[16] (chloroquine resistance transporter) which causes an efflux of chloroquine from the food vacuoles. PfCRT is dependent on Ca2+ channels, and calcium channel blockers like verapamil reversed the resistance to chloroquine. Some of these resistance reversers tried are verapamil, desipramine, trifluperazine and chlorpheniramine.[17]

Newer drugs

Presently, ACTs form the first-line treatment for falciparum malaria as resistance to other drugs is widespread. Now, signs of artemisinin resistance have started to appear along the Thai–Cambodian border.[18] Chloroquine resistance also started in this area and spread to other parts of the world. The artesunate-resistant strains had increased parasite clearance time when compared to susceptible strains.[19] Whether the resistance is restricted to artemisinin derivatives alone or extends also to the entire class of endoperoxide antimalarials is not known.[20] This poses a serious problem and development of new drugs is of top priority now. The drugs in preclinical, phase I and phase II of drug development are listed in Table 1.[2122]

Table 1

Antimalarials in clinical development

Phase III drugs

Arterolane and piperaquine

Arterolane is a synthetic ozonide developed by Ranbaxy. Though it has the endoperoxide moiety, it differs structurally from artemisinins and so it is believed to be effective against artemisinin-resistant strains. In the multicentric phase II trials, the parasite clearance time was found to be 12.8 and 12.6 h for 100 and 200 mg, respectively.[23] The combination of arterolane and piperaquine is undergoing phase III trials in India, Thailand and Bangladesh. The combination is developed as a once daily therapy for 3 days.[24]

Azithromycin and chloroquine

In high malaria transmission areas, WHO recommends the Intermittent Preventive Treatment of malaria in pregnancy (IPTp) with at least two courses of sulfadoxine (500 mg) and pyrimethamine (25 mg) during second and third trimesters and at least three courses in case of HIV positive women.[4] As resistance to sulfadoxine-pyrimethamine is increasing, a new combination of azithromycin and chloroquine is tried in IPTp.[25] Both azithromycin and chloroquine are found to be safe in pregnancy. Azithromycin targets the 70S ribosomal subunit of apicoplast and hinders polypeptide development. An additive action is seen when used in chloroquine-resistant strains. In chloroquine-resistant strains, a synergistic action is seen and there is a reversal of chloroquine resistance. Azithromycin is a p-glycoprotein substrate and can inhibit the p-glycoprotein mediated efflux and this can probably cause the reversal of chloroquine resistance.

Artesunate and pyronaridine

The combination of artesunate and pyronaridine has completed phase III trials and is awaiting approval from the regulatory agencies. It is found to be noninferior to artemether–lumefantrine in uncomplicated P. falciparum malaria.[26]

Newer drug targets[2728]

Food vacuole

Hemoglobin is broken down into heme and is converted to hemozoin in the food vacuole. This pathway has been targeted by the currently available aminoquinolones. Falcipains and plasmepsin proteases which break down hemoglobin are now considered as potential targets for new antimalarial agents.

Parasitophorous vacuole

The malarial parasite lies in a parasitophorous vacuole (PV) inside the RBCs, which is surrounded by a parasitophorous vacuole membrane (PVM). This is an active membrane with protein trafficking occurring between the parasite, RBC cytoplasm and RBC membrane. After maturing, the merozoites are released by the lysis of the PVM by the action of proteases. Among these, some subtilisin like proteases are targeted for drug development.

Protein export

The proteins destined for the RBC cytoplasm and membrane have specific recognition sequences. These recognition sequences are revealed in the endoplasmic reticulum by the action of a protease identified as Plasmepsin V. If these proteases can be inhibited, it will disrupt the cellular protein trafficking and seriously impair the development of malarial parasite.

Apicloplast

Apicoplast is an organelle which is synonymous to chloroplasts in plant cells and has a bacterial origin. It has its own transcription and translation machineries and is involved in the biosynthesis of many isoprenoids, fatty acids and proteins. Fosmidomycin acts by inhibiting the isoprenoid biosynthesis pathway and is in phase II trials.

Mitochondria

The electron transport chain of mitochondria is a potential drug target and atovaquone blocks the mitochondrial respiration.

Nucleoside biosynthesis

Dihydroorotate dehydrogenase involved in pyrimidine biosynthesis is targeted.

ERADICATION OF MALARIA

The Bill and Melinda Gates Foundation announced a road map for the eradication of malaria in October 2007.[29] It was soon followed by support from the WHO and the Roll Back Malaria Partnership. A Global Malaria Action Plan[30] was formulated by the Roll Back Malaria Partnership in 2008 which had the following goals:

Reduce malaria cases by 50% in 2010 and 75% in 2015;

Reduce malaria deaths by 50% in 2010 and near zero preventable deaths in 2015 from 2000 levels and

Eradication of malaria.

The key factors proposed for eradicating malaria are as follows:

Reducing malaria burden;

Malarial vaccine and

Vector control.

Reducing malaria burden

The immediate goal of the eradication programs is to reduce the malarial burden, thereby reducing the transmission of the disease. It mainly consists of scaling up of existing tools like using antimalarial drugs for treating every case of malaria, use of insecticides to reduce vectors, using insecticide treated/long-lasting insecticide impregnated bed nets to reduce the incidence of the disease.

Malarial vaccine

Malaria vaccines form the cornerstone of malaria eradication as humans are the only intermediate hosts. Though primate hosts are identified, transmission from them is rare and occurs only at few places. Most of the vaccines developed are against P. falciparum as it causes the majority of deaths. The vaccines developed are basically of three types:[31]

Pre-erythrocytic stage vaccine,

Blood stage vaccine and

Transmission blocking vaccine.

SPf-66

SPf-66 was the first malaria vaccine against P. falciparum that was tried in clinical trials in the 1990s. It is a synthetic hybrid chimera containing three merozoite protein-derived peptides and a sporozoite repeat sequence intercalated twice, developed by Manuel E. Patarroyo. The initial studies in South America showed promising results, but the later studies in Africa showed reduced efficacy. A Cochrane review has found no evidence for protection against infections.[32] It is now tried with a new adjuvant QS-21 for improving its immunogenicity.

RTS,S

It is a pre-erythrocytic stage vaccine against P. falciparum, which inhibits the parasite entry into liver cells. It is the most successful vaccine candidate, currently in phase III trials. It is a hybrid molecule expressed in Saccharomyces cerevisiae, consisting of

Tandem repeat tetrapeptide (R),

C-terminal T-cell epitope containing (T) regions of CSP,

Hepatitis B surface antigen (S) and

Unfused S antigen (S).

It is formulated with either one of two adjuvants:

ASO2: Oil in water emulsion containing immunostimulants monophosphoryl lipid A (MPL) and QS-21, a fraction of Quillaia saponaria or

ASO1: Liposomal formulation replaces the oil in water emulsion.

It induces both humoral and cell-mediated immunity against the P. falciparum parasite. Phase II studies in children aged 1-4 years after a 45-month follow-up showed a vaccine efficacy of 30.5% against a first or only episode of clinical malaria, 25.6% against all episodes and 38.3% against severe malaria.[33] The prevalence of P. falciparum in the vaccinated group was 34% lower compared to the control group. If the phase III trials turn out to be successful, it will be launched in 2012.

The other vaccines currently under development are listed in Table 2.[34]

Table 2

Malarial vaccines under development

Basically, the pre-erythrocytic vaccines are targeted against the circumsporozoite protein (CSP) and the blood stage vaccines are directed against the merozoite surface protein (MSP) and apical membrane protein (AMP).

Challenges facing vaccine development[31]

Though malarial vaccines may provide a panacea for malaria, a number of factors hamper the development of vaccines. Some of them are as follows:

Inadequate understanding of host mediators of immunity

Lack of surrogate markers in clinical trials

Only few antigens are tried as vaccine candidates

Only few immune enhancing adjuvants are used with the vaccines

Most importantly, funding for the development of vaccines is inadequate. To state the facts, the R&D funding for malaria was US$ 468.5 million, with only US$ 88.4 million for vaccine development in 2007 in comparison to US$ 1.08 billion funding for HIV/AIDS, with US$ 692 million for vaccine development.

Vector control

The conventional approaches for vector control consisted of using insecticides, larvivorous fish, environmental engineering, etc. But these approaches are not very effective in controlling the vector population due to a variety of reasons like development of resistance to the insecticides, inability to create and maintain environmental sanitation in developing countries, etc. So, some novel approaches are pursued for controlling the vector menace. Some of these novel techniques are discussed below.

Sterile insect technique

This involves making the male mosquitoes sterile by exposing them to γ radiation from a 60Co or 137Cs source or chemosterilants like aziridinyl compounds and alkylating agents. The female Anopheles mosquitoes mate only once in their lifetime, and by making them to mate with the sterile male mosquitoes by releasing these sterile mosquitoes in large numbers, their reproduction can be greatly reduced. This technique has been successfully employed for elimination of New World screw-worm fly in North and Central America.[35] The sterile insect technique has been assessed in field trials with Culex quinquefasciatus and Aedes aegypti in New Delhi around 1971 and in El Salvador with Anopheles albimanus.[35]

Transgenic techniques

The radiation used in sterile insect techniques not only affects the germ cell lines but also deleteriously affects the somatic cells which can reduce the competitiveness of the released sterile mosquitoes to mate.[36] This reduced competitiveness of the male mosquitoes can be overcome by transgenic techniques in which a dominant conditional lethal factor can be expressed in the male mosquitoes.[37] In this Release of Insects carrying a Dominant Lethal (RIDL)[38] technique, the released mosquitoes on mating transfer the lethal gene to the progeny and its expression will lead to embryonic lethality. This technique is employed in producing A. aegypti mosquitoes which can pass the lethal factor to the offsprings, causing embryonic lethality.[39] A pilot field trial with release of transgenic A. aegypti has been approved in Malaysia.[40] Another approach will be to express the dominant lethal gene only in the female mosquitoes. The males will carry the dominant lethal gene, but will not express them and they transmit it to the eggs. The eggs developing into female mosquitoes will express the lethal gene and will be destroyed and only male progeny will be produced.[37] This will reduce the population of female mosquitoes, which will lead to a decline in the mosquito population.

Another technique involves expressing hemolytic lectins in transgenic mosquitoes which inhibit the development of ookinete[41] in the stomach of the mosquitoes, thereby blocking the sexual stage of the malarial parasite.

SUMMARY

With 243 million cases and 863,000 deaths worldwide, malaria is an important public health issue. Presently, artemisinin based combination therapy forms the first-line of treatment for malaria as resistance to other antimalarials is widespread. Signs of artemisinin resistance are seen at some areas and pose a threat for the control of malaria. Most of the new drugs in late stages of development have the endoperoxide active moiety and whether they will be active against the resistant strains is not known. Malarial vaccines offer hope for the eradication of malaria with the RTS,S vaccine expected to be launched in 2012. Though the conventional methods of vector control are still followed, some novel approaches are tried for effective vector control. Eradication of malaria has been only a dream until recently and it is turning into reality because of effective antimalarials, vaccines and novel vector control strategies.