Alternative First-line Malaria Treatment

Abstract

Malaria is a disease affecting millions of people, especially in Africa, Asia, and South America, and has become a substantial economic burden. Because malaria is contracted through the bite of a mosquito vector, it is very challenging to prevent. Bed nets and insect repellents are used in some homes; others do not have or use them even when available. Thus, treatment measures are crucial to controlling this disease. Artemisinin-based combination therapy (ACT) is currently the first-line treatment for malaria. ACT has been used for decades, but recently, there has been evidence of potential resistance. This threat of resistance has led to the search for possible alternatives to ACT. In sub-Saharan Africa, Azadirachta indica, or simply neem, is a plant used to treat a variety of ailments, including malaria. Neem is effective against one of the more deadly malaria parasites Plasmodium falciparum. Reports show that neem inhibits microgametogenesis of P. falciparum and interferes with the parasite’s ookinete development. Although there is substantial in vitro research on the biological activity of A. indica (neem), there is limited in vivo research. Herein, we discuss the in vivo effects of neem on malaria parasites. With A. indica, the future of malaria treatment is promising, especially for high-risk patients, but further research and clinical trials are required to confirm its biological activity.

INTRODUCTION

About half of the world’s population is at risk of malaria.[1] In tropical countries such as Ghana and Nigeria, malaria is hyperendemic. In fact, malaria is the second leading cause of death among Ghanaian adults (after HIV/AIDS).[2] Malaria is caused by the parasitic protozoan Plasmodium and is transmitted by female Anopheles mosquitoes.[2] In Ghana, the most harmful species of the Plasmodium parasite is Plasmodium falciparum. This parasite has infected the majority of Ghanaians who contract malaria. The clinical signs of malaria include fever, chills, anemia, delirium, and possibly even multi-organ system failure.[2]

Historically, chloroquine has been an effective form of malaria chemotherapy since 1946.[1] However, the parasite has become resistant to many of the conventional drugs, including chloroquine and the sulfadoxine–pyrimethamine combinations.[3] Currently, the most effective chemotherapy is artemisinin-containing (or artemisinin-based combination) therapies, commonly known as artemisinin-based combination therapy (ACT). Once artemisinin is combined with other antimalarial drugs, the therapy becomes much more effective than the use of a single antimalarial. The artemisinins not only kill malaria parasites, but they also inhibit the parasites’ major metabolic processes including glycolysis and synthesis of nucleic acids and protein.[4] In addition, artemisinins inhibit the development of gametocytes, the stage of the parasite that transmits Plasmodium from human to mosquito.[4] Although artemisinins are excellent antimalarial drugs, ACT therapy has its drawbacks. ACT is expensive and not easily accessible to a number of West Africans, including Ghanaians, especially those in rural areas. It is difficult for many Ghanaians to seek care for malaria due to financial factors and limited access to healthcare facilities. For these reasons, many Ghanaians in both rural and urban environments use traditional medicine to treat their medical ailments.[2]

In addition, many clinically important drugs come from herbal sources. In fact, artemisinin originates from qinghao, a Chinese herb.[4] Since previously successful antimalarial drugs such as quinine and artemisinin come from plants, many researchers believe that further research on promising plants such as Azadirachta indica may lead to novel plant-derived antimalarial drugs. The herbal market has become a key component of Ghana’s health-care system. Antimalarials derived from herbal plants, which are common in Ghana, may make it easier for Ghanaians to access antimalarial therapy.

Although native to the Indian subcontinent, the neem tree is now well established in Ghana due to its distribution by Indian migrants.[5] Currently, to treat malaria as well as other illnesses, many Ghanaians boil the leaves of neem and drink it as tea. The effectiveness of A. indica (the neem tree extract) against malaria has been studied.[1] A 1997 experimental study demonstrated “…that A. indica is highly effective against chloroquine-resistant and chloroquine susceptible sexual and asexual stages of human malaria P. falciparum.”[1] It was also shown that other extracts from neem, such as nimbolide and gedunin have antimalarial activity which was potent against P. falciparum.[6]

Herbal antimalarial drugs continue to be used in Ghana due to their availability, effectiveness, and affordability. Comparatively, while it would cost approximately $20–$100 to treat acute malaria with current therapies, the average cost of treating malaria using herbal remedies ranges from $1 to $2.[3]

SCIENTIFIC CONCEPTS

One characteristic of malaria that makes it difficult to control is its dual reproductive life cycle involving two hosts. An understanding of the malaria life cycle shows why ACT is a successful antimalarial therapy and how neem could become a more affordable and accessible first-line alternative in sub-Saharan Africa. We briefly describe the life cycle of P. falciparum below.

Life cycle of Plasmodium

Four human malaria parasites exist: Plasmodium ovale, Plasmodium vivax, Plasmodium malariae, and P. falciparum. P. falciparum is a malignant malaria parasite that accounts for most of the malaria mortality rates.[1] The life cycle of malaria parasites, as shown in Figure 1, is seemingly complex. The parasite has both sexual and asexual reproduction. Sexual reproduction occurs in the female blood-feeding Anopheles mosquito, which acts as a definitive and primary host in the parasite’s life cycle. Male gametes fertilize female gametes in the gut of the mosquito, producing zygotes.[7] The zygotes become ookinetes, which then migrate to the outer wall of the mosquito’s midgut, where they mature into oocysts. The oocysts grow, and sporozoites form within them. The oocysts then rupture, releasing the sporozoites, which then circulate to the mosquito’s salivary glands. The malaria life cycle continues when the mosquito inoculates the sporozoites into a human, the intermediate host.[7]

Figure 1.

Life cycle of the malaria parasite including asexual stage in human and sexual stage in mosquito.[1] (Adapted from Awofeso et al., 2011)

When the blood-feeding Anopheles mosquito bites the human, the sporozoites circulate to the liver. After penetrating the liver cells, the sporozoites transform and multiply to produce thousands of merozoites.[8] Following this replication stage in the liver, the merozoites undergo asexual reproduction in the erythrocytes.[7] Each asexual merozoite invades a red blood cell using its apical complex. Once inside the cell, the parasite makes the surface of the erythrocyte sticky. This cytoadherence results in sequestration of the infected red blood cells, with resultant consequences that manifest as severe falciparum malaria.[9] In addition, inside the erythrocyte, Plasmodium develops within a membrane-bound vacuole first as a trophozoite and later as a schizont through multiple nuclear divisions (schizogony).[9] The schizont matures, bursting the erythrocyte, consequently releasing a number of merozoites that will invade more erythrocytes.[8] This blood cycle is responsible for the clinical symptoms of malaria. Some of these invading merozoites may develop into nonreplicating sexual gametocytes but instead represent the transmission stages of the parasite. If a mosquito takes up these gametocytes, gametes from the male gametocyte may fertilize gametes from the female gametocyte forming zygotes, which as mentioned earlier, can give rise to infective sporozoites.[8]

Artemisinin-based combination therapy treatment of malaria

In ACTs, artemisinin is the main component of the therapy because it is effective against resistant malaria parasites.[4] Artemisinin is extracted from qinghao, a traditional Chinese herb, and its derivatives were made to enhance its solubility in oil and water.[10] Artesunate, the water-soluble derivative of artemisinin, can be administered orally, intramuscularly, intravenously, or intrarectally.[10] The World Health Organization adopted ACTs as the first choice for malaria treatment.[11] Specifically, the combination of artemisinin with a variety of companion drugs increases the effectiveness of malaria treatment.[11] Artemisinin and its derivatives work on the late-stage ring parasites and trophozoites. The structure of artemisinin includes C-O-O-C, an endoperoxide bridge that is absent from other antimalarials.[12] This bridge is a source of oxygen-free radicals, which mediate the killing of the parasites. In general, free-radical-generating drugs produce reactive oxygen species (ROS), including hydroxyl radical and superoxide, which in turn cause oxidative damage to the cell. Artemisinin, on the other hand, does not create large numbers of ROS. In actuality, artemisinin becomes a free radical through a reaction catalyzed by iron.[12] However, besides cost, another major disadvantage of artemisinin is its short half-life, because it is only briefly effective in plasma.[12]

Azadirachta indica

The neem extract A. indica contains about 135 compounds.[1,13] The compounds that exhibit antimalarial activity include nimbolide, gedunin, and azadirachtin (AZA).[1,13] Within the general class of active compounds of neem (triterpenes) is the most effective group of compounds called limonoids, for which azadirachtin (AZA) is a member as seen in Figure 2.[13] A. indica is steadily becoming established as a mosquito larvicidal agent, insect repellent, and chemotherapy for malaria infection.[1] In one study in particular, Adebayo and Krettli. found that extracts of neem seeds killed all the erythrocytic stages of P. falciparum.[14] Thus, indicating that neem attacks the asexual stages of P. falciparum, which include the ring-stage parasites found in the human. In addition, neem exhibits a strong effect against the sexual stages (mature gametocytes) of P. falciparum. Consequently, the action of neem is different from that of artemisinin because artemisinin only affects the immature stages of the parasite.[14] Gametocytes correspond to the transmission stage of malaria. Thus, further investigation into the effectiveness of neem against the mature stages of the parasite would also be beneficial. Notwithstanding, researchers may consider the use of a combination of neem extracts with artemisinin to allow for different modes of action against malaria. Moreover, if the parasite were to become resistant to one drug, the other companion drugs could still be effective in killing the resistant parasite.

Figure 2.

Molecular structure of the limonoid azadirachtin.[13] (Adapted from Drabu et al. 2012)

ANTIMALARIAL STUDIES OF AZADIRACHTA INDICA

Antimalarial activity of A. indica has been explored in vitro, demonstrating the gametocytocidal activity of the extract as well as its efficacy against chloroquine-resistant strains of the malaria parasite.[6] A recent in vivo rodent experiment supports the value of A. indica.[6]

In vitro inhibitory activity of Azadirachta indica

Gametocytocidal activity on Plasmodium falciparum

The inhibitory activity of A. indica has been explored in vitro. Specifically, one study demonstrated that A. indica can “…eliminate more than 90% of P. falciparum immature and mature gametocytes in culture, at a concentration of 2.5 µg/mL.”[6] In addition, the neem tree extract inhibits microgametogenesis and cytoskeletal functions. The AZA impedes maturation of P. falciparum and P. berghei gametes through a process called exflagellation.[6] This impediment induces “…an interruption of the endomitotic divisions and the formation of rigid extensions on axonemes, preventing their motility.”[6] In addition to preventing motility of the parasites, AZA most likely “…disrupts cytoplasmic and axonemal microtuble organization…by compromising the functionality of the microtubule organizing centres.”[6]

Efficacy against chloroquine-resistant strains of Plasmodium falciparum

Chloroquine is one of the main drugs that P. falciparum is resistant to. Fortunately, in vitro studies have shown that A. indica is effective against chloroquine-resistant strains of P. falciparum. Specifically, gedunin and nimbolide are the most active molecules of A. indica.[6]

In vivo activity of Azadirachta indica

Rodent malaria experimental model

In vivo inhibitory effect of AZA on P. falciparum has yet to be explored. A recent study examines the in vivo transmission-blocking potential of AZA using a rodent malaria model. The model used was a P. berghei/Anopheles stephensi/BALB/c mouse. Although the model used P. berghei, it was validated as beneficial for determining the potential of neem compounds as agents for blocking the transmission of P. falciparum.[6] For the experiment, NeemAzal (NA), a standardized extract of A. indica seed kernels, containing limonoids was inserted into the experimental mice as intraperitoneal inoculations.[6] The mice were also gametocytaemic, meaning they contained the gametocytes of the parasite. In the experiment, female mosquitoes ingested the sexual stages (gametocytes) of P. berghei by taking a blood meal on the NA-treated gametocytaemia mice. The impacts of NA as well as AZA tech grade on oocyst maturation were evaluated. To do this, untreated mosquitoes were fed a second blood meal on neem-treated mice. After mosquito infection on day 10, oocyst development was examined in dissected mosquitoes. Figure 3 shows the three categories the authors used to evaluate the oocysts.[6]

Figure 3.

Images (light microscope × 400) of Plasmodium berghei oocyst development stages on day 10 following mosquito infection. (a) Immature oocyst, before formation of sporoblasts. (b1 and b2) Immature oocysts, with sporoblasts as well as budding sporozoites. (c) Mature oocyst, with fully developed sporozoites.[6] (Adapted from Lucantoni et al. 2010)

Inhibition of microgametogenesis of Plasmodium falciparum

This experiment resulted in blockage of sporogonic development in the mosquitoes that ingested the blood meal on the NA-treated gametocytemic mice. In fact, within the group of mice treated with NA at 50 mg/kg body weight, none of the 138 mosquitoes examined in three experiments demonstrated the presence of oocysts.[6] This experimental result, as displayed in Figure 4, was confirmed by exposing healthy mice to bites of the mosquitoes that fed on the NA-treated mice. These healthy mice failed to develop Plasmodium infections.[6] The interference of ookinete development shows that microgametogenesis was inhibited. The importance of this finding lies in the fact that the development of gametocytes signifies the eventual transmission of malaria.

Figure 4.

Effects of neem products on Plasmodium berghei zygote and ookinete stage (sexual stages in midgut).[6] (a) Samples prepared 18 hours after Anopheles stephensi females ingested infected blood meal. (b) 20 hours after ingestion of infected blood meal. NA25, NeemAzal^® 25 mg/kg; NA50, NeemAzal^® 50 mg/kg; AZA50, azadirachtin 50 mg/kg. Please see Lucantoni et al. 2010. (Adapted from Lucantoni et al. 2010)

Interference with ookinete formation

The experiment also suggests that the NA treatment interferes with ookinete development. Since the treated mosquitoes were unable to produce oocysts, the mosquitoes were unable to infect healthy mice. Specifically, the examination of mosquito midgut slides after the infective blood meal demonstrated that “…NA activity was directed to the early sporogonic stages: The total number of zygotes and postzygotic forms was reduced in the NA-treated group compared to controls.”[6] This finding indicates that the NA interferes with the development of the parasite before zygote formation. To identify the alterations of the postzygotic stages of the parasite, the researchers examined Giemsa-stained midgut smears of NA 50 mg/kg treated mosquitoes. The Giemsa stain revealed morphological alterations of the postzygotic forms of these mosquitoes. Figure 5 compares these morphological changes to the control mosquitoes. In the article, Lucantoni et al. stated that: “The irregular cell shape, the jagged cell membrane, the inability to form the typically strongly stained apical complex and to perform the elongation process are compatible with an interference with the microtubule organizing processes involved in cytoskeletal and organelle rearrangements.”[6]

Figure 5.

Images (light microscope ×1000) of Giemsa stained P. berghei zygotes and ookinetes from midgut of Anopheles stephensi, 18–20 h following infective blood meal. (a-h) Control mosquitoes, (i-t) NeemAzal 50 mg/kg treated mosquitoes.[6] (Adapted from Lucantoni et al. 2010)

MEDICAL OR TECHNOLOGICAL AREAS RELATED TO THE RESEARCH

For years chloroquine was the drug of choice to treat malaria. In the mid-20^th century chloroquine resistance emerged. As a result, ACT became the drug of choice, as it is effective against chloroquine-resistant parasites. Unfortunately, recent studies describe the potential rise in artemisinin resistance.[14] A. indica may possibly be able to combat this type of resistance.

Challenges with current treatment of Plasmodium falciparum malaria

Chloroquine resistance

One of the major reasons for the high malaria-related mortality is the incidence of P. falciparum and its resistance to commonly used drugs such as chloroquine.[14] Chloroquine resistance originated in Southeast Asia and South America in the 1960s and spread to countries with endemic malaria.[15] It was suggested that chloroquine resistance came to be because chloroquine-resistant parasites accumulate less chloroquine in their acidic food vacuoles than the chloroquine-sensitive parasites.[15] Another possible reason for the decrease in the accumulation of chloroquine in chloroquine-resistant parasites is an increase in the vacuolar pH of these parasites. This increase in pH could have been due to inhibition of a proton pump resulting in a weakened mechanism of vacuolar acidification.[15] Finally, another possible reason for chloroquine resistance is the loss or modification of a protein in the parasite that is involved in the uptake of chloroquine.[15]

Artemisinin resistance

Recently, there has been a rise in artemisinin resistance which could be devastating to global malarial control.[16] A group of researchers examined P. falciparum susceptibility and molecular markers of artemisinin resistance in Pailin, western Cambodia, and Wang Pha, northwestern Thailand.[16] Their findings showed that compared to northwestern Thailand, P. falciparum had reduced in vivo susceptibility to artesunate in western Cambodia.[16] “Artesunate resistance was characterized by a markedly prolonged time to parasite clearance, with relatively little heterogeneity among patients.”[16] Unfortunately, it is unclear the exact contributions of resistance to artemisinin, although in recent years, western Cambodia has demonstrated a decline in the efficacy of ACT.[16] Thus, it is becoming increasingly important to prevent and limit the spread of these artemisinin-resistant parasites from western Cambodia.

Need for Azadirachta indica to combat resistance

Consequently, A. indica is a likely possibility for a new, alternative treatment for malaria. Another limitation of ACT is that after treatment, gametocytaemia reduction is quite a slow process, specifically lasting three to 4 weeks. In fact, membrane feeding assays demonstrated that on the 14^th day following the start of treatment, around 60% of children treated with artemether–lumefantrine and 40% of children treated with sulfadoxine–pyrimethamine (SP) plus artesunate sulfadoxine were infective to mosquitoes.[6]

APPLICATION AND CURRENT STUDIES

Use of Azadirachta indica in high-risk populations

Effects of Azadirachta indica on pregnant women

Pregnant women represent one of the populations at high risk of contracting malaria. In fact, malaria infection is not only more harmful to the pregnant woman, but it also has serious effects on the fetus or even the newborn.[17] Because the immunity of pregnant women is altered, they are more susceptible to severe and complicated malaria.[17] In addition, pregnant women who contract malaria are likely to have worsened anemia, which can increase the risk of maternal death.[18] The challenge with treating malaria in pregnant women arises from the fact that the antimalarial drugs can end up being teratogenic (harmful to the developing fetus). Thus, another problem facing malarial treatment in pregnant women is determining which is riskier – the chance of teratogenicity of the antimalarial drugs or the risk of undertreatment.[11] There are several ways to reduce the redundant exposure to antimalarials by the mother and fetus. Specifically, treatment occurs “…after parasitological confirmation by expert microscopy or…following a rapid diagnostic immunochromatographic antigen detection test.”[11] At times, however, it is difficult to clinically diagnose malaria in pregnant women because there tends to be an overlap between the symptoms of nonsevere malaria and “normal” pregnancy symptoms.[18] In some parts of sub-Saharan Africa, intermittent preventive treatment of malaria in pregnancy is a treatment whereby pregnant women (despite whether or not they have malaria) are given at least two full curative doses of SP during pregnancy.[17]

Many pregnant women, among others, use herbal medicine to treat their ailments, including malaria. Many adults use herbal malaria remedies to avoid the expenses of biomedicine and transport to medical care. In particular, to treat general illnesses in central Ghana, pregnant women commonly use nonbiomedical remedies to guarantee the safe delivery of a healthy baby.[18] A recent study demonstrated that pregnant women in northern Ghana do not use neem leaves to treat their malaria because of the bitter taste of the leaves and because the neem was seen as a source of miscarriage.[18] On the other hand, a recent study investigated the positive effects of consuming A. indica during pregnancy by examining rats. The pregnant rats treated with aqueous leaf extract of A. indica demonstrated increased packed cell volume, hemoglobin concentration, and a number of white and red blood cells.[19] This increase in hematological parameters shows the potential for A. indica to improve the burden of anemia in pregnancy exacerbated by malaria in pregnancy. One of the possible reasons for this increase in blood parameters could be the hematopoietic properties of flavonoids and quercetin, which are components of the aqueous extract.[19] In addition, the increase in white blood cell count could be attributed to A. indica boosting a macrophage response, stimulating the lymphatic system to produce more white blood cells. Although these effects of A. indica in pregnant rats who have malaria seem promising, further research into the use of neem leaves in pregnant women later in pregnancy should be conducted.

Effects of Azadirachta indica on children

Children represent another group of people at high risk for malaria. Cerebral malaria is a severe form of malaria that primarily affects children. Those who survive cerebral malaria exhibit neurological problems, including damaged consciousness, seizures, and even coma.[20] Consequently, cerebral malaria is a major source of neurodisability among children in areas endemic to malaria.[20] Since A. indica is a commonly used plant for the treatment of malaria, one study, in particular, explores the potential of A. indica to treat cerebral malaria by examining the effects of an A. indica ethanolic extract on mice.[20] Although the extract did not protect the experimented mice from death, the extract did indeed demonstrate neuroprotective effects that were most likely due to alleviating the edema and adjusting the triggers of neurological apoptosis.[20] For example, the findings suggested that A. indica protected pyramidal neurons from cerebral malaria-induced apoptosis.[20] These results show that further research should be conducted to create new drugs to fight cerebral malaria, especially in children.

Effects of Azadirachta indica on malaria patients with HIV/AIDS

HIV/AIDS patients are also at high risk of contracting malaria. The main problem this group of people face is dealing with two treatment paradigms: The first as participants of the protocol of HIV care through biomedicine and the second as self-care through nonbiomedical means.[21] A recent qualitative study examining this population in northeast Tanzania demonstrated that patients find it difficult to manage HIV and malaria concurrently. For instance, some found themselves adjusting the timing of taking the different HIV and malaria drugs to deal with the possible “friction” between these strong drugs.[21] Some patients noted that when they took the anti-retrovirals (ARVs) and antimalarials simultaneously, they experienced fatigue and exhaustion.[21] Patients suffering from HIV and malaria also face a pill burden. In the Tanzanian study, one patient described her situation in which she had to take nine tablets a day, which was so much that she even wanted to stop taking them.[21]

For the aforementioned reasons, the neem tree can also be beneficial to HIV/AIDS patients with malaria. Antiretroviral activity is another positive effect of A. indica, among many others. One study presents evidence of this activity by evaluating an acetone-water neem leaf extract in patients with HIV/AIDS.[22] As a result of the study, the neem extract not only inhibited adhesion of the malaria-infected red blood cells, but it also inhibited HIV from invading human lymphocytes.[22] The experiment also resulted in an increase in CD⁴+ cell count, bodyweight, and hemoglobin.[22] Therefore, the neem tree could be a plausible solution to the pill burden, among the other issues facing HIV patients with malaria. This is because the A. indica can concomitantly serve as an antimalarial and antiretroviral.

CONCLUSION

Although the use of herbal remedies to treat malaria in sub-Saharan Africa is far from a new concept, robust research and clinical trials on this subject are limited. Research on neem tree extracts still has a long way to go, considering the few in vivo studies. However, the evidence from the in vitro studies as well as the in vivo rodent studies are promising for the future use of A. indica as a malarial therapeutic agent. Not only does neem rapidly kill the schizonts and gametocytes of P. falciparum, but it also is a low-cost alternative to ACT.[2]

Additionally, many West African countries already use herbal medicines, including neem, to treat their ailments. Thus, further research would be beneficial to examine the mode of action and bioactive molecules of A. indica and the target stage of the parasite’s life cycle. Future bioassay-guided fractionation can be done to identify and assess the biological activity of neem extracts against both the asexual and sexual stages of different species of Plasmodium.[6] Because A. indica contains various components with antimalarial efficacy, the production of an antimalarial combining the most potent and active components of the neem tree will surely increase the transmission-blocking efficacy along with the therapeutic efficacy. In addition, the presence of multiple bioactive components might reduce the potential of falciparum resistance against neem. Thus, it has become increasingly crucial that A. indica continue to be researched and scientifically validated because it may be the solution to the endemic problem of malaria in sub-Saharan Africa.

Financial support and sponsorship

Support was received from Mercer University School of Medicine for this study.

Conflicts of interest

There are no conflicts of interest.