1.1 THE BURDEN OF MALARIA
Malaria is a mosquito-borne infectious disease of humans and other animals caused by parasitic protozoans (a type of single cell microorganism) of the Plasmodium type (World Health Organization, 2014).It is the fifth cause of death from infectious diseases worldwide (after respiratory infections, HIV/AIDS, diarrhea diseases, and tuberculosis) (World Health Organization, 2002). Malaria ranks second as killer disease in Africa, after the recent epidemic of Ebola viral disease (World Health Organisation, 2014) and HIV/AIDS. Symptoms include fever, fatigue, vomiting and headaches. Seizures, coma or death can occur in severe cases. These symptoms usually begin ten to fifteen days after being bitten by the vector.
Malaria is transmitted by the bite of an infected female Anopheles mosquito. The bite introduces the parasites from the mosquito's saliva into a person's blood, and then the parasite travels to the liver where they mature and reproduce (Caraballo, 2014). Five species of Plasmodium can infect and be spread by humans. The disease can recur months later in those who have not been treated properly. In those who have recently survived an infection, re-infection typically causes milder symptoms. Globally, malaria causes about over two million deaths annually, one every 15 to 30 seconds, killing mainly children under five years of age in Sub-Saharan Africa (Breman et al., 2007). About 90% of malaria deaths in the world today occur in Africa, south of the Sahara due to the fact that most of the infections in Africa are caused by Plasmodium falciparum, the most dangerous of all the malaria parasites. Increasing resistance of Plasmodium falciparum to antimalarial drugs and the Anopheles vector to insecticides presents great challenges to those battling this scourge (Breman et al., 2007).
1.2 EPIDEMIOLOGY OF MALARIA
Recent estimates have shown that as many as 3.3 billion people live in areas at risk of malaria in 109 countries globally (World Health Organization, 2008). In Africa, all malaria endemic have about 25-40% of outpatient clinics visits are for malaria. Between 20-50% of all hospital admissions are consequences of malaria in this same countries. Malaria is a contributor to death among hospital impatients with high case-fertility rates due to late presentations unavailability of effective drugs and inadequate management. This high burden is due to misdiagnosis because many facilities lack laboratory capacity and it is often difficult clinically to distinguish malaria from infectious diseases (World Health Organisation, 2003).
1.3 CONSEQUENCES OF MALARIA INFECTION
1.3.1 Morbidity and mortality in children and pregnant women
Most children experience their first malaria infection during the first year or two of life, when acquired adequate clinical immunity has not been acquired, which makes these early years particularly dangerous. About 90% of all malaria deaths in Africa occur in children. In areas of stable transmission, adult women have high level of immunity which can be impaired in the first pregnancy with the result that rise of infection increases (Murphy et al., 2001).The lack of acquired immunity makes infants and young children very vulnerable to malaria. Most cases of severe malarial anemia and deaths occur in infants and young children in areas of intense malaria transmission. Pregnant women are also at high risk of malaria. Approximately 50 million women living in malaria endemic countries throughout the world become pregnant each year (Snow, 2000).
In stable transmission areas, the major effect is malaria-related anaemia in the mother and presence of parasites in the placenta resulting in low-birth weight which contributes substantially to child deaths. Pregnant women have little or no immunity to malaria and their risk of developing severe disease as a result of malaria infection is two to three times higher than that of non-pregnant women living in the same area (Luxemburger, 1997) in unstable transmission settings. Consequently, malaria during pregnancy contributes to maternal deaths in both stable and unstable transmission areas.
There are three major ways by which malaria can contribute to death in children. Firstly, overwhelming acute infection which frequently presents as seizures or comas which can kill directly or indirectly. Second, repeated infections contribute to the development of severe anemia which can substantially increases the risk of deaths. Third, malaria infections make young children more susceptible to other common childhood illnesses, such as diarrhea and respiratory infections which can contribute to mortality (Molineaux, 1997).
1.3.2 Socio-economic burden of malaria
Malaria puts a heavy economic burden on endemic countries and contributes to the cycle of poverty people face in many countries. For example, it is estimated to have in Africa alone contemporaneous costs of at least US$12 billion per year in direct losses (e.g. illness, treatment, premature death), but many times more than that in lost economic growth (Gallup et al., 2001). Poor people are at an increased risk of becoming infected with malaria more frequently. Child mortality rates are known to be very high in poorer households and a substantial proportion of these deaths is due to malaria. In a demographic surveillance system in rural areas in Africa, under-5 mortality following acute fever is about 39% higher in the poorest socio-economic group than the richest (Mwageni, 2002).
Malaria is responsible for a high proportion of public health expenditure on curative treatment, and substantial reductions in malaria incidence can free up available health resources and facilities and health workers time to tackle other health problems (World Health Organisation, 2003). Poor families live in dwellings that offer very little protection against mosquitoes and are less able to afford insecticide-treated nets. They are also less likely to be able to pay either for effective malaria treatment or for transportation to a health facility capable of treating this disease.
1.4 PREVALAENCE OF MALARIA INFECTION
Four Plasmodia species infect human beings: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium Ovale. Plasmodium falciparum and Plasmodium. vivax cause the significant most of malaria infections. Plasmodium. falciparum, which causes most of the severe cases and deaths, is mostly found in tropical regions, such as the sub-Saharan Africa and Southeast Asia, as well as in the Western Pacific and in countries which share the Amazon rainforest (Snow et al., 1999). Plasmodium.vivax generally is mostly common in most of Asia (especially Southeast Asia) and the Eastern Mediterranean, and in most endemic countries of the Americas. Plasmodium.malariae and Plasmodium. ovale contribute to a small number of malaria infections. Plasmodium.ovale is found in Africa and sporadically in Southeast Asia and the Western Pacific. Plasmodium.malariae has a similar geographical distribution to Plasmodium falciparum but its incidence is underestimated (World Health Organisation, 1999).
Estimates have been shown by the World Health Organization that in 2010 there were 219 million cases of malaria resulting in 660,000 deaths (Nadjm et al., 2012). Other estimations have shown that the number of cases at between 350 and 550 million for falciparum malaria and deaths at about 1.24 million in 2010 up from 1.0 million deaths in 1990. The majority of cases (65%) occur in children that are under 15 years (Murray et al., 2012). About 125 million pregnant women are at risk of infection in Sub-Saharan Africa and up to 200,000 estimated deaths are associated with maternal malaria every year.
There are about 10,000 malaria cases per year in Western Europe, and 13001500 in the United States (Taylor et al., 2012). About 900 deaths have been known to occur in Europe between 1993 and 2003. The global incidence of disease and resulting mortality has also been known to decline in recent years. According to the World Health Organization, deaths attributable to malaria in 2010 have been reduced by over a third from a 2000 estimate of 985,000, largely due to the widespread use of insecticide-treated nets and artemisinin-based combination therapies (Howitt et al., 2012).
Malaria is presently endemic in a broad band around the equator, in areas of the Americas, many parts of Asia, much of Africa and in Sub-Saharan Africa, 8590% of malaria fatalities occur (Layne, 2007). An estimate for 2009 has reported that countries with the highest death rate per 100,000 of population were Ivory Coast (86.15), Angola (56.93) and Burkina Faso (50.66) (Provost, 2011). A 2010 estimate also indicated the deadliest countries per population were Burkina Faso, Mozambique and Mali. As of 2010, about 100 countries have endemic malaria according to the World Health Organization. Every year, 125 million international travellers visit these countries, and more than 30,000 eventually contract the disease (Kajfasz, 2009).
The geographic distribution of malaria within large regions is complex, and malaria-afflicted and malaria-free areas are often found close to each other (Greenwood et al., 2002). Malaria is prevalent in tropical and subtropical regions due to high rainfall, consistent high temperatures, high humidity and stagnant water in which mosquito larvae readily mature, this provides them with the environment they require for continuous breeding. Malaria is very common in rural areas than in cities. For example, several cities in the Greater Mekong Sub-region of Southeast Asia are malaria-free, but this disease is prevalent in many rural regions, including along international borders and forest fringes (Yang et al., 2012).
In contrast, malaria is present in Africa in both the rural and urban areas, with lower risk in the larger cities (Machault et al., 2011). The Sγo Paulo municipality and nearby areas in southeastern Brazil are considered non-endemic regions. In 2013, 146 autochthonous cases of malaria were identified in Sγo Paulo State, which represents 22% of the total occurring outside the Amazon region. There was also a 256% increase compared to 2006 (Marques et al., 2008). Approximately 60% of malaria cases in the Americas occur in Brazil (Costa et al., 2012) which exclusively occurs in the Amazon Region. Some regions of Brazil outside of the Amazon Region receive patients presenting with malaria, either from the Amazon Region or from other countries which are mainly Latin American and African nations. Malaria is a major health problem in Brazil, with 177,783 cases registered in the Amazon Region as at 2013 (more than 99% of Brazilian cases occur in the Amazon Region) (Joao et al., 2014).
1.5 STRATEGIES FOR CONTROL OF MALARIA
Malaria can be controlled in various ways which include: malaria chemotherapy, vector control, insecticide treated nets, mosquito elimination, prevention of bites and to an extent, vaccines. The presence of malaria in an area requires a combination of high human population density, high anopheles mosquito population density and also high rates of transmission from humans to mosquitoes and from mosquitoes to humans (World Health Organisation, 2010). If any of these is lowered sufficiently, the parasite can eventually be eliminated from that area, such as in North America, Europe and parts of the Middle East. However, if the parasite is eliminated from the whole world, it could become re-established if conditions revert to a combination that favors the parasite's reproduction (World Health Organisation, 2014).
Prevention of malaria can be more cost-effective than treatment of the disease in the long run, the costs required are out of reach of many of the world's poorest people and there is also a wide difference in the costs of control (i.e. maintenance of low endemicity) and elimination programs between countries (Sabot et al., 2010). An example is China, whose government in was able to pursue malaria elimination in the Chinese provinces which required a small expenditure on health. In contrast, a similar program in Tanzania can cost an estimated one-fifth of the public health budget (Tang et al., 2010)
1.5.1 Chemotherapy for malaria
Malaria chemotherapy can be used for different reasons and are thus classified as:
220.127.116.11 Radical cure: It is the process of eliminating the dormant liver forms of malaria parasite (hypnozoites) once the parasite has been eliminated from the blood stream. Drugs such as primaquine (Baird et al., 2003) can be used to clear the dormant forms of these parasites. Tafenoquine is also another anti-malarial that is used as a potential treatment for malaria and malaria prevention (Shanks et al., 2001). It is used in the treatment of hypnozoite stages of Plasmodium falciparum and other plasmodium species such as Plasmodium vivax and Plasmodium ovale. An advantage of Tafenoquine is that it has a half-life of about 2-3 weeks which may be sufficient to clear hypnozoites.
18.104.22.168 Suppressive treatment and prophylaxis: This is a preventive treatment for malaria. It is recommended for prevention of malaria infection in travelers visiting endemic areas (Jacquerioz et al., 2009). The protective effect does not start immediately, and people visiting areas where malaria exists usually start taking the drugs one to two weeks before arriving and then continue taking them for four weeks after leaving. Drugs such as atovaquone/proguanil are taken two days before travel and continued for seven days afterward (Freedman, 2008). Preventative drugs are used only for a short-term by visitors and travellers which is due to the cost of the drugs, side effects from long-term use, and the difficulty in obtaining anti-malarial drugs outside of wealthy nations (Fernanado et al., 2011).
Suppressive prophylactics such as chloroquine, proguanil, mefloquine and doxycycline are also effective at killing the malaria parasite once it has entered the erythrocytic stage (blood stage) of its life cycle (Jacquerioz et al., 2009) and have no effect until the liver stage is complete (Freedman, 2008). It is therefore recommended to continue to take suppressive prophylactics four weeks after leaving the area of risk. Most of these drugs are also used in treatment sometimes. Chloroquine may be used where there is parasite sensitivity (Croft et al., 2009). Due to Plasmodium resistant to one or more medications, mefloquine, doxycycline, or the combination of atovaquone and proguanil hydrochloride (Malarone), can be used frequently needed.
22.214.171.124 Clinical cure: This involves clearance of asexual parasites and clinical symptoms in patients. The World Health Organization recommends the use of artemisinin-based combination therapy (ACT) for the treatment of malaria in countries experiencing resistance to anti-malarial drug monotherapy. Artemisinin derivatives are known for rapid reduction of parasite biomass (White, 1998). These classes of drugs have very short half-lives and their use alone in monotherapy is associated with a high percentage of treatment failure. Artemisinins are conventionally used in combination with slower acting anti-malarial drugs in order to improve the cure rate of infections responding inadequately to mono-therapy and possibly to prevent or delay the emergence of resistance (World Health Organization, 2010). Artemisinin combination treatments such dihydroartemisininpiperaquine (Artekin) combination, can be used to treat resistant Plasmodium falciparum in Asian countries (Liwang et al., 2009).
Artemisinin combination therapies with a 3-day regimen have been shown to be very effective and useful through numerous clinical trials. A fixed-dose artesunatepyronaridine combination has also shown excellent efficacy against uncomplicated falciparum malaria in children in other clinical trials (Schreier et al., 2008). In addition, several other ACTs such as artesunateamodiaquine, artesunatesulfadoxinepyrimethamine (SF) and artesunatechlorprogunanildapsone have been developed and have proved to be effective. In most areas, artemisinin combination therapies are highly effective against falciparum malaria, with cure rates exceeding 90% (White, 2008).
1.5.2 Vector Control
Methods used to decrease malaria by reducing the levels of transmission by mosquitoes are referred to as vector control. The most effective insect repellents are based on DEET (diethyltoluamide) (Kajfasz, 2009). Insecticide-treated mosquito nets (ITNs) and indoor residual spraying (IRS) are very effective in preventing malaria among children in endemic areas (Lengeler, 2004). Quick treatment of confirmed cases with artemisinin-based combination therapies (ACTs) can also reduce transmission (Palmer, 2006). Mosquito nets are also useful in keeping mosquitoes away from people and reducing infection rates and transmission. Nets are are often treated with an insecticide designed to kill the mosquito before it can find a way past the net. Insecticide-treated nets are estimated to be twice as effective as untreated nets and offer greater than seventy percent protection compared with no nets (Reddy et al., 2011). Studies have shown that the use of insecticide treated nets saved the lives of an estimated 250,000 infants in Sub-Saharan Africa between 2000 and 2008 (Howiit et al., 2012) and about 13% of households in Sub-Saharan countries own insecticide treated nets.
An estimate of about 1.7million (1.8%) African children living in areas of the world where malaria is common have been protected by insecticide treated nets in 2000. This number increased to 20.3 million (18.5%) African children using ITNs in 2007, leaving 89.6 million children unprotected (Noor et al., 2009). An increased percentage of African households (31%) are estimated to own at least one insecticide treated net in 2008. Indoor residual spraying which involves the spraying of insecticides on the walls of the home is also very effective in killing mosquitoes before they can bite another person and transfer the malaria parasite (Enayati, 2010). An issue with all forms of Insecticide treated net is insecticide resistance. Mosquitoes affected by insecticide treated nets tend to rest and live indoors, and due to irritation caused by spraying, they tend to rest and live outdoors, meaning that they are less affected by the indoor residual spraying (Pates et al., 2005).
Other methods that can be used to decrease malaria transmission include, decrease mosquito larva by decreasing the availability of open water in which they develop or by adding substances to decrease their development can be effective in some locations (Tusting et al., 2013). Community participation and health education strategies can promote awareness of malaria and the importance of control measures in order to reduce the incidence of malaria in disease from becoming fatal (Lallo, 2006). Intermittent preventive therapy can also be applied to successfully control malaria in pregnant women, infants and in preschool children in areas where transmission is seasonal (Donegan et al., 2012).
1.5.3 Malaria vaccines
Parasite vaccines generally face the challenge of generating immunity with an immunogen that reflects only a tiny fraction (less than 1%) of the composition of the organism, a challenge that has been met only rarely in vaccinology (Lightowlers, 2010). Natural immunity to malaria develops in most residents of endemic areas, which generally takes some years of exposure and is imperfect. Extensive immuno-epidemiological studies provide limited insight into what the best antigens to include in a vaccine might be: natural immunity predominantly targets a wide variety of blood-stage antigens and no one antigen has appeared to be especially important in providing protection (Mash et al., 2006).
Most malaria antigens which have been selected as vaccine candidates are the targets of natural immunity and exhibit significant genetic polymorphism, and a key blood-stage antigen, P. falciparum erythrocyte membrane protein-1 (PfEMP1) (Andrian, 2011).
126.96.36.199 Transmission blocking vaccines
Anti-malarial transmission-blocking vaccines (TBVs) tend to inhibit the transmission of Plasmodium from humans to mosquitoes by targeting the sexual/ookinete stages of the parasite. The use of such interventions can subsequently result in reduced cases of malarial infection within a human population, leading to local elimination (Sala et al., 2015).
188.8.131.52 Pre-erythrocytic vaccines
Pre-erythrocytic malaria vaccines stop injected sporozoites from reaching the liver or to direct cellular immune responses towards eliminating infected hepatocytes. These vaccines help to prevent or clear liver stage infections. They also cause induced immune response which eliminates sporozoites after injection (Elike et al., 2014).
184.108.40.206 Blood stage vaccines
Blood stage vaccines can be used to prevent against this stage of the parasite life cycle which would prevent or reduce severity and complications of the disease (Good et al., 2002). Blood stage vaccines are targets formerozoite surface proteins which play important roles in the initial recognition and attachment of merozoites to the red blood cell surface (Fujioka et al., 1999). As proteins on the merozoite surface are exposed to the host immune system, they are thought to be targets of immune responses. Antibodies against merozoite surface proteins neutralize the parasite by agglutinating or opsonizing merozoites, thereby preventing RBC recognition and invasion, or affecting growth of the new intracellular parasite within the RBC (Holder et al., 1999)
1.6 CHALLENGES FOR MALARIA ELIMINATION AND CONTROL
Malaria continues to be a significant public health issue and a major hindrance to economic growth. Management of malaria and sustained control presents significant challenges. Effective interventions include; widespread implementation of effective vector control measures such as long lasting insecticide treated bed nets, indoor residual spraying prompt and effective treatment with artemisinin combination therapies following accurate diagnosis (Kokwaro, 2009). Reducing malaria transmission by reducing gametocyte carriage with effective drugs can be an important factor in highly endemic areas.
The provision of an appropriate formulation of artemisinin combination therapies for infants and young children, who bear the greatest burden of malaria, presents a particular challenge. Administering anti-malarials to infants and small children can be very difficult, stressful, and time consuming (Ogutu et al., 2008). Crushing of malaria tablets and mixing with food or water to ease administration to young children, and the bitter taste can cause children to spit out the crushed tablets and, thereby not receiving an optimal dose that will cure the malaria infection. Storage of suspensions, syrups and accurated dosing of syrups may be difficult as it requires precise volume measurement in the field (Abdulla et al., 2008). Education of healthcare workers and patients about the prevention and treatment of malaria is another challenge in the management of the disease. Another difficulty in reducing the toll of malaria is reaching remote communities with poor transport systems, and achieving timely reordering to maintain supplies of artemisinin combination therapies (World Health Organisation, 2008).
One of the greatest difficulties in reducing the toll of malaria is drug resistance in P. falciparum which has developed to all antimalarials including artemisinin drugs. ACTs reduced the incidence of drug resistance in many areas however, resistance to artemisinin and ACTs have been reported in areas of south-east Asia (Dondorp et al., 2009). Solving the problems and addressing the continuing challenges presented by malaria in the years ahead will require responsive strategies such as innovative vector control methods, widespread implementation of biological diagnosis prior to treatment with effective antimalarials, promoting compliance and preserving effectiveness of artemisinin combination therapies, development of newer antimalarials and effective vaccines (Kokwaro, 2009).
Artemisinin derivatives are currently the worlds most potent anti-malarial drugs. Recently, resistance emerged to these drugs in south-east Asia (Dondorp et al., 2009; Ariey et al., 2014; World Health Organisation, 2014).This poses a threat to the control and eradication of malaria. Artemisinin and its derivatives provide faster clearance of parasites than any other anti-malarial drug and these drugs are part of frontline combination therapies in areas where drug resistant Plasmodium falciparum exists. Clinical evidence for artemisinin resistance in south-east Asia was first reported in 2008 and was subsequently confirmed by a detailed study from western Cambodia (Phyo et al., 2012). Resistance in neighboring Thailand was reported in 2012 in Northern Cambodia, Vietnam and Eastern Myanmar in 2014 (Ashley et al., 2014). Emerging resistance was reported in Southern Laos, central Myanmar and North-Eastern Cambodia in 2014. The parasite's kelch gene on chromosome 13 appears to be a reliable molecular marker for clinical resistance in southeast Asia (Amaratunga et al., 2014; Ariey et al.,2014). Laboratory works have also shown ring stage parasites enter a dormant stage after exposure to artemisinins (Teuscher et al., 2012; Mok et al., 2011).
Malaria control methods which include insecticide-treated bed nets, and other preventive measures are important in the control of malaria, in the absence of a licensed vaccine and acquired, fully-protective immunity, chemotherapy has been and continues to be one of the best ways to prevent deaths, to control symptoms, and to eliminate parasites from a given geographic region. A recurrent problem with chemotherapy is that parasites, like other microbes, can and will rapidly evolve mechanisms to escape drug pressure and survive. Although arguably augmented by other factors, such as reduced spending on malaria control, the emergence and spread of multidrug-resistant P. falciparum parasites has probably contributed, directly or indirectly, to hundreds of millions of new cases each year and to millions of unnecessary deaths between 1970 and 2000.
Malaria continues to be an important public health problem globally, the emerging trend of increasing resistance to antimalarials will increase malaria burden in endemic areas and worldwide. Very few newer antimalarials are being developed, thus strategies to preserve the efficacy and use of artemisinins could reduce the impending burden of malaria in endemic areas if artemisinin resistance spreads to such areas. Emerging artemisinin resistance provides an impetus for studies into newer non-antimalarial drugs that potentiate artemisinin drugs to help improve their efficacy, reverse parasite insensitivity or prolong their usefulness in malaria chemotherapy.
The activity of artemisinins can be investigated in vitro as well as in vivo. The in vitro systems generally use Plasmodium falciparum with infected human erythrocyte while in vivo models mainly use rodents infected with Plasmodium berghei. Recently, a modified method that can be used to evaluate the ex-vivo activity of artemisinins was developed and validated (Witkowski et al., 2014). The ring stage survival assay (RSA) enables stage-specific evaluation of the activity of artemisinin drugs to determine inherent resistance associated with ring stage asexual parasites. It is imperative to evaluate using this inexpensive method to determine susceptibility of parasite isolates to determine early if there is emerging resistance to artemisinin.
1.8 AIM OF THE STUDY
The broad aim of the study is to determine susceptibility of P. falciparum isolates to artemisinin derivatives and potentiating effects of ketoconazole on asexual parasites.
1.8.1 Objectives of study
(i) To determine ex-vivo stage specific effects of artemisinin derivatives on Plasmodium falciparum isolates from Ota southwest Nigeria.
(ii) To determine Plasmodium falciparum stage susceptibility and parasite dynamics by fluorescent and molecular methods.
(iii) To determine potentiating effects of non-antimalarials combinations with artemisinin drugs.