**Francesco Mancini 1,\* and Benedetto Nastasi 2**

Received: 26 June 2019; Accepted: 18 July 2019; Published: 19 July 2019

**Abstract:** Electrification of the built environment is foreseen as a main driver for energy transition for more effective, electric renewable capacity firming. Direct and on-time use of electricity is the best way to integrate them, but the current energy demand of residential building stock is often mainly fuel-based. Switching from fuel to electric-driven heating systems could play a key role. Yet, it implies modifications in the building stock due to the change in the temperature of the supplied heat by new heat pumps compared to existing boilers and in power demand to the electricity meter. Conventional energy retrofitting scenarios are usually evaluated in terms of cost-effective energy saving, while the effects on the electrification and flexibility are neglected. In this paper, the improvement of the building envelope and the installations of electric-driven space heating and domestic hot water production systems is analyzed for 419 dwellings. The dwellings database was built by means of a survey among the students attending the Faculty of Architecture at Sapienza University of Rome. A set of key performance indicators were selected for energy and environmental performance. The changes in the energy flexibility led to the viable participation of all the dwellings to a demand response programme.

**Keywords:** energy flexibility; retrofitting interventions; residential consumption; demand response; electrification in the built environment


**3. Conventional medicines**

Malaria is a serious and potentially life threatening disease. It can lead to fatal outcomes in only few days, thus treatment should be started as soon as possible. According to their chemical structure and activity, the available antimalarial agents are grouped into 5 classes as shown in **Table 1** [54, 55]. The key targets of modern antimalarial agents are asexual blood stages of *Plasmodium* species (**Figure 2**), responsible for the malaria symptoms [56]. The *4- aminoquinolines* are blood schizonticidal agents and their mechanism of action is ascribed to their ability to form drug-heme adducts and accumulation of free heme, which is toxic for the parasite [57]. It is also belived that their mode of action is attributed by their inhibition of hemoglobin endocytosis and digestion or disruption of normal vesicle trafficking [58].

**Chloroquine**, the prototype anti-malarial drug, is the drug of choice for both treatment and chemoprophylaxis of all malaria parasites except for chloroquineresistant *Plasmodium* strains. In addition to schizonticidal activity, it is also moderately effective against gametocytes of *vivax, ovale,* and *malariae* but not against those of *falciparum* gametocytes*.* Chloroquine does not eliminate dormant liver forms of *vivax* and *ovale,* for that reason primaquine must be added for the radical cure of these species. Although almost all strains of *malariae* are susceptible, *falciparum*, *vivax* and even some *ovale* strains have been reported as resistant to chloroquine. It is no longer recommended for prophylaxis against *falciparum* [59]. Although chloroquine is the first-line therapy for *vivax* malaria in majority of endemic countries, resistance is the core problem facing this drug in different parts of the world. In Africa and South America, its resistance to *falciparum* first appeared in 1978 and 1996, respectively [60, 61]. In Ethiopia, chloroquine treatment failure against *falciparum* and *vivax* malaria was reported for the fist time from Debre Zeit in 1995. Then after, chloroquine resistance has abeen detected

#### **Table 1.** *Classification of antimalarial drugs.*

*Malaria: Introductory Concepts, Resistance Issues and Current Medicines DOI: http://dx.doi.org/10.5772/intechopen.98725*

#### **Figure 2.**

*Plasmodium life cycle with phases targeted by antimalarial drugs. 4-Aminoquinolines target the parasite at the stage where hemoglobin is degraded by parasite protease enzymes. 4-aminoquinolines such as chloroquine and amodiaquine have no effect on the pre-eythrocytic liver stages of parasite development.*

in Ethiopia [25, 62]. However, drug resistance to chloroquine can be reversed by certain agents, including verapamil, desipramine, and chlorpheniramine, but the clinical value of resistance-reversing drugs is not established [59].

**Amodiaquine** is closely related to chloroquine and it probably shares mechanisms of action and resistance with chloroquine. Amodiaquine has been widely used to treat malaria (10 mg base/kg/day for 3 days) because of its low cost, limited toxicity, and, in some areas, effectiveness against chloroquine-resistant strains of *falciparum.* The most important current use is in combination therapy with: (i) sulfadoxine-pyrimethamine (SP) for prophylaxis, and (ii) artesunate [artemisininbased combination therapy (ACT)] for treatment [13, 59]. **Piperaquine** is a potent and well-tolerated bisquinoline compound thought to act like chloroquine. This lipophilic drug is rapidly absorbed and has an excellent activity on chloroquineresistant species. Currently, piperaquine combined with dihydroartemisinin (DHA) in co-formulated tablets has shown remarkable efficacy and safety in treating *falciparum* malaria, without visible drug resistance. Piperaquine has a larger halflife (28 days) than amodiaquine (14 days), mefloquine (14 days), and lumefantrine (4 days), leading to a prolonged duration of post-treatment prophylaxis with DHApiperaquine than with other ACTs; this characteristic is advantageous especially in high transmission areas. DHA-piperaquine (one of the ACTs) is used to treat uncomplicated malaria [59, 63].

*Eight-aminoquinolines*, tissue schizonticidal agents, are belongs to the only class proven to be effective against the hypnozoites (exoerythrocytic forms) of *vivax* and *ovale* (**Figure 2**)*.*  Although *falciparum* gametocyte clearance takes days, gametocytes are sterilized within hours; therefore, its effect on oocyst and sporozoite formation (and thus onward transmission of treated infection) precedes its effect on gametocytes carriage. Due to this effect some literatures classify primaquine as sporontocide. The addition of primaquine single dose to ACT is, therefore, recommended by the WHO to reduce gametocyte burden and thus transmission. It has weak activity against the asexual blood stage of *vivax* malaria but with negligible activity against *falciparum* malaria [13].

**Primaquine** (the prototype drug in 8-aminoquinolines) is indicated for radical cure of *vivax* or *ovale* malaria; for presumptive anti-relapse therapy (terminal prophylaxis- use after the completion of travel to an endemic area to markedly diminish the hypnozoite stages) in population widely exposed to *vivax* or *ovale;* to decrease onward *falciparum* malaria transmission in *falciparum* malaria elimination programmes and in areas threatened by *falciparum* resistance to artemisinins; and as an option for primary (causal and suppressive) prophylaxis against all *Plasmodium* species. Except its use in primary prophylaxis (prevent establishment of infection in the liver by inhibiting the pre-erythrocytic schizogony), primaquine is used in conjunction with an effective blood schizonticide (either ACT or chloroquine) to eradicate erythrocytic stages of *vivax* or *ovale* malaria, and to reduce the possibility of emerging drug resistance [13]. Its mechanism of action is unknown but it is thought to interfere with the cellular respiration of the parasite by means of generating oxygen-free radicals and deregulating the electron transport [64].

**Quinine** is one of the four antimalarial cinchona alkaloids and has rapid schizonticidal activity against intraerythrocytic malaria parasites. Quinine kills large ring and tropozoite asexual parasites and is gametocidal against *vivax, ovale* and *malariae* but not *falciparum* malaria [65]. Its mechanism of action has not been completely elucidated. The most widely accepted hypothesis is that the drug can inhibit hemozoin crystallization interfering with the heme detoxification process inside the food vacuole (membrane enclosed cell vacuole with a digestive function) [66]. The antimalarial and resistance mechanism of quinine is thought to share similarities to chloroquine.

**Mefloquine** was first used to treat chloroquine-resistant *falciparum* malaria in Thailand. However, the slow elimination of mefloquine fostered the emergence of drug-resistant parasites [63]. This drug is structurally related to quinine and has two racemic forms, *erythro-* and *threo-*, each composed of a pair of enantiomers, of which the racemic mixture of the *erythro-* enantiomers is the most active against Plasmodia [13]. Mefloquine is a blood schizonticide, active against the erythrocytic stages (15 mg/kg in a single dose) of all malaria parasites. It has more or less the same stage specificity of action as quinine, killing mainly the large ring and trophozoite asexual parasites. It has no significant pre-erythrocytic activity. In combination with artesunate, it can be used to treat uncomplicated malaria [13, 66]. The drug is especially useful as a chemoprophylactic agent for travelers spending weeks, months, or years in areas where *falciparum* and *vivax* infections are endemic due to its slow elimination (delayed half-life), except in clearly defined Thai border regions associated with MDR strains. The mechanism of action is still unknown, probably being different from 4-aminoquinolines. Activity on the parasite seems to be related to the ability of mefloquine to interfere with the transport of hemoglobin from the erythrocyte to the food vacuole. It is also proposed that it inhibits endocytosis of the cytosol by the parasite [13, 63].

**Halofantrine hydrochloride** is a phenanthrene methanol structurally related to quinine. It is effective against erythrocytic (but not other) stages of all four human malaria species. This synthetic anti-malarial is effective against MDR (including mefloquine resistant) *falciparum* malaria, but its use is limited by irregular absorption and cardiac toxicity. It should not be used for chemoprophylaxis. The mechanism of action by halofantrine is mysterious. It may be similar to that of chloroquine, quinine, and mefloquine; through forming toxic complexes with ferritoporphyrin IX that damage the membrane of the parasite [59, 67].

**Antifolates** are drugs that target two important enzymes of the folate pathway, namely the Dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS). Proguanil and Pyrimethamine target DHFR, whereas sulfadoxine and dapsone act on DHPS. Pyrimethamine and proguanil are active against susceptible

#### *Malaria: Introductory Concepts, Resistance Issues and Current Medicines DOI: http://dx.doi.org/10.5772/intechopen.98725*

strains of all four human malaria species. In *falciparum* malaria sensitive antimalarial drugs, proguanil exhibit activity against both the primary hepatic stages and the asexual blood stages, thus sufficiently controlling the acute attack and usually eradicating the infection. Chloroguanide (proguanil) is also effective in treating acute *vivax* malaria, but relapses may arise after the drug is withdrawn because the latent tissue stages are not affected by this drug. Proguanil therapy does not obliterate gametocytes, but acts as a sporontocide (oocytes in the mosquito gut fail to develop normally) and thus ablate the transmission. It is not actually used alone as resistance to proguanil develops very quickly. Proguanil accentuates the mitochondrial membrane-potential-collapsing action of atovaquone against *falciparum* but displays no such activity by itself. The combination of proguanil and atovaquone is known as Malarone® and it is used as chemoprophylaxis in adults and children ≥11 kg. Atovaquone-proguanil may be considered for the treatment of uncomplicated malaria in travelers (adults and children ≥5 kg) outside malaria-endemic areas. It is highly effective and safe in a 3-day regimen for treating mild-to-moderate attacks of chloroquine- or SP-resistant *falciparum* malaria. The combination of chloroquine (500 mg weekly) and proguanil (200 mg daily) was previously widely used, but with increasing resistance to both agents it is no longer recommended [59, 63].

Pyrimethamine (2, 4-diaminopyrimidines) is a slow-acting blood schizontocide with antimalarial effect ssimilar to proguanil. However, pyrimethamine has greater antimalarial potency. The effectiveness of pyrimethamine against liver stages of *falciparum* is less than that of proguanil, and at therapeutic concentrations, pyrimethamine fails to eradicate hypnozoites of *vivax* and gametocytes of any malaria species. It raises the number of circulating mature infecting gametocytes of *falciparum*, likely leading to increased transmission to mosquitoes during treatment period. Pyrimethamine is typically administered with either a sulfonamide such as sulfadoxine or sulfone such as dapsone to enhance its antifolate activity. Sulfonamides and sulfones are weakly active against erythrocytic schizonts but not against liver stages or gametocytes. They are not used alone as antimalarials but are effective in combination with other agents. Although, no longer recommended due to drug resistance, pyrimethamine was used in synergistic combination with sulfadoxine (Fansidar®) or sulfalene (Metakelfin®) for treatment of uncomplicated malaria and with dapsone for prophylaxis. SP is active predominantly against later development stages of asexual parasites. In the few areas in which it remains effective, SP can be used with artesunate for the treatment of acute uncomplicated malaria. Its resistance is caused by point mutations in DHPS and DHFR [13, 63].

**Atovaquone**, a highly lipophilic analogue of ubiquinone, is active against all *Plasmodium* species, *Pneumocystis jiroveci* and *Toxoplasma gondii*. It is highly active against asexual blood stage (erythrocytic schizonts) of *falciparum* malaria. This drug (only administered orally) is also effective against liver stages (tissue schizonts) of *falciparum* (allowing prophylaxis to be discontinued only one week after the end of exposure) but not against *vivax* hypnozoites. Since atovaquone is tissue schizonticidal, malarone has an advantage over mefloquine and doxycycline in requiring shorter periods of treatment before and after the period at risk for malaria transmission, but it is more expensive than the other agents. Atovaquone selectively inhibits the parasite mitochondrial electron transport chain at the cytochrome bc1 complex. Selectivity is due to structural differences between the cytochrome b encoded by the parasite mitochondrial DNA and that encoded by the host mitochondrial DNA. Regeneration of ubiquinone (electron acceptor for *Plasmodium* dihydroorotate dehydrogenase [DHODH] enzyme, essential for pyrimidine biosynthesis) is the primary function of mitochondrial electron transport in *falciparum* species. Synergism activity between proguanil and atovaquone is resulted from the

capability of non-metabolized proguanil to enhance the mitochondrial toxicity by atovaquone [59, 63, 68].

**Artemisinins** (endoperoxide sesquiterpene lactone) is a potent and fast acting blood schizonticidal killing all parasite stages, inducing more rapid parasite clearance and fever resolution than any other currently licensed antimalarial drug. Artemisinins have no effect on hepatic stages. They have been reported to reduce gametocytogenesis (young *falciparum* gametocytes), thus reducing transmission of malaria (preventing the spread of resistant strains). However, artemisinin has some pharmacokinetic limitations such as low solubility, poor bioavailability, and short half-life. To overcome some of these problems, semi-synthetic derivatives have been developed. First generation derivatives include the oil-soluble methyl ether, artemether (artemotil [arteether] is a closely related compound); the water soluble hemi-succinate derivative, artesunate; and DHA. Moreover, all active compounds possess a distinctive 1,2,4-trioxane pharmacophore, which is essential for the antimalarial activity since the corresponding acyclic compounds lacking the endoperoxide are biologically inactive [67, 69]. The precise mechanism of action of artemisinin is unclear and still controversial [70]. It has been suggested that the endoperoxide bond undergoes reductive activation by iron2+ or iron3+-heme. This redox reaction produces carbon-centered radicals that alkylate target molecules leading to parasite's death [71]. Alternative views suggest that artemisinin inhibits *P. falciparum* encoded sarcoendoplasmic reticulum Ca2+-ATPase (*PfATP6*) [72]. Another proposed mechanism is that artemisinins act as oxidant drugs through oxidation of flavin adenine dinucleotide (FADH2) and parasite flavoenzymes [73].

The standard treatment of malaria employs ACTs to increase treatment efficacy and reduce selection pressure for the emergence of drug resistance. Artemisinins cause a significant reduction of the parasite burden. As such, only 6–8 days of treatment are required to remove the parasites from the blood. Artemisinins do not display significant clinical cross-resistance with other drugs. Artemisinins should not be used for chemoprophylaxis because of their short half-life, which translates into high recrudescence rates. ACTs have lower toxicity and are considered safe to use in children and non-pregnant mothers. However, the widespread distribution of counterfeit (clinically sub-standard) agents that contain small quantities of artemisinin derivative threatens the effective administration of ACTs. The artemisinins and its derivatives generally are not used alone because of their limited ability to eradicate infection completely or its short plasma t1/2 translates into substantial treatment failure rates. ACT consists of an artemisinin derivative combined with a long-acting antimalarial drug. To promote patient adherence to treatment by reducing course of therapy from 6 to 8 days to 3 days and to avoid the use of artemisinins as monotherapies due to their brief duration of action, fixed-dose combination formulations into a single tablet are available for all recommended ACTs (artemether + lumefantrine, artesunate + amodiaquine, artesunate + mefloquine, dihydroartemisinin + piperaquine, pyronaridine + artesunate, and artesunate + SP), except for artesunate plus SP. Artesunate-SP is not recommended in many areas owing to unacceptable levels of resistance to sulfadoxine-pyrimethamine. **Lumefantrine** is a fluorene derivative belongs to the group of quinine, halofantrine and mefloquine. This drug is belived to act similar to other members of the group (prevent haem detoxification within the food vacuole of the parasite, thus causing accumulation of the toxic haem complex). Lumefantrine (benflumetol) is formulated with artemether (COARTEM) [13, 63].

The WHO recommends ACT for the treatment of uncomplicated malaria caused by *falciparum* parasite or by chloroquine resistant *vivax*, *ovale, malariae* and *knowlsi*. Quinine plus clindamycin is used for uncomplicated malaria treatment in the first trimester of pregnancy [13]. In Ethiopia, Coartem (artemether-lumefantrine) is

#### *Malaria: Introductory Concepts, Resistance Issues and Current Medicines DOI: http://dx.doi.org/10.5772/intechopen.98725*

suggested as the first-line drug for uncomplicated *falciparum* malaria and chloroquine for other species (*vivax, malariae, ovale* and *knowlesi*) but oral quinine is considered as a second option [62]. Given their rapid and potent activity against even MDR parasites, injectable artesunate becomes the drug of choice for severe malaria globally in infants, children, lactating women and pregnant mothers of all trimester. After one day, the course of therapy should be completed using oral ACT [13]. Quinidine plus tetracycline, doxycycline, or clindamycin is the treatment of choice for severe malaria in the USA [74]. In Ethiopia, injectable artesunate is the drug of choice and intramuscular artemether is an alternative agent. When these 2 drugs are not available, injectable quinine is used to treat severe malaria [62].

Co-resistance of quinine with artesunate-amodiaquine (one of the most widely used ACTs) was fully verified both *in vivo* and *in vitro*. Given the widespread use of ACT worldwide, the suggestion that ART pressure might also favor quinine resistance is of major concern. Undeniably, the present dependence on artemisinins to manage both uncomplicated and complicated malaria, together with absence of possible therapeutic options, leaves decision-makers with very limited alternatives. This would have very bad consequences not only in the therapy of individual cases, but would cripple efforts to conquer malaria globally [75]. According to the current study, DHA-piperaquine is not treating malaria effectively across the eastern Greater Mekong subregion. A highly drug-resistant *falciparum* co-lineage is evolving, acquiring novel resistance mechanisms. So, resistance among artemisinin and its partner drug will continue to evolve, producing *Plasmodium* strains more capable of surviving treatment, which can subsequently spread across a wider geographical area. As a consequence, accelerated *falciparum* malaria elimination in this region is required urgently, to avert further spread and avoid a potential global health emergency. In the dearth of new antimalarial classes to replace the present firstline therapies, the use of existing treatments in the form of triple ACTs, in which an artemisinin is combined with 2 partner agents such as DHA-piperaquine and mefloquine, could be a viable alternative [76].

Despite decades of intense research, no licensed malaria vaccines are available until now [77]. A lot, but a better understanding is required on host immunity and the *Plasmodium* to improve vaccines. In Phase-3 clinical testing, the first proven antiparasite vaccine (a circumsporozoite protein vaccine [RTS, S/AS01]) reduced clinical malaria in children. Nonetheless, young infants do not respond well, and implementation studies with mortality endpoints are awaited. The irradiated *P. falciparum* sporozoites such as PfSPZ, which is closer to pivotal Phase-III trials, can be manufactured and have been shown to prevent infection in some African countries. Most recently, African trials of gamete protein vaccines started and placental malaria vaccines entered human testing. Blood-stage targets of protective antibodies remain unknown, but new proteins implicated in erythrocyte invasion and egress offer promise [78]. Limitations in efficacy, absence of standard predictive biomarkers of protective efficacy and the need to constantly update vaccine formulations due to antigenic polymorphism further underscore the current reliance on chemotherapy [79]. However, the occurrence of resistance (malaria parasites survive and/or multiply despite the proper administration and absorption of an antimalarial medicine in the dose normally recommended) [80] among commonly used drugs is a major problem. Resistance against antimalarial drug results in a global revival of malaria creating a major problem to malaria control. Indiscriminate and widespread utilization of antimalarial agents contributes to *Plasmodium* species to evolve and develop resistance mechanisms [81, 82]. As a result, old and novel chemicals are under per-clinical and clinical studies. Despite the widespread development of resistance and difficulties in poor areas to afford and access effective antimalarial drugs, currently used and potent drugs, such as

artemether, chloroquine and quinine, are obtained from plant sources. Hence, it is imperative to focus on traditionally used medicinal plants for the discovery of possible new innovative antimalarial sources for the future.

### **4. Genetic basis of drug resistance**

Resistance to antimalarial compounds occurs because of the parasites selection with genetic mutations such as single nucleotide polymorphisms (SNP) or gene amplifications that confer decreased susceptibility [83]. A number of factors aid the emergence of current antimalarial drug resistance. Some of them, among others, are the mutation rate of *Plasmodium*, the overall parasite load, the strength of drug selected, the treatment compliance, and poor adherence to treatment guidelines. Inappropriate dose, poor pharmacokinetic profile, fake drugs lead to inadequate drug exposure on parasites [84, 85], and poor quality antimalarial (falsified antimalarial without active ingredients) drugs may aid and abet the occurnce of resistance by increasing the risk of hyperparasitaemia, recrudescence, and hypergametocyopaenia [86, 87].

The two malaria parasites (*falciparum* and *vivax*) that cause most of malaria cases of human beings have developed resistance to almost all current antimalarial drugs. The capability of these *Plasmodium* species to develop resistance is mainly due to the large numbers of parasites in the infected individual's bloodstream at the time of the asexual blood stage infection in conjunction with the mutability of their genomes [88]. Now a day, controlling MDR *falciparum* malaria is become a very challenging work for the reason that endogenous allelic exchanges occurred in *falciparum* species have increased the treatment failures and drastically increased the resistance level globally. Since evolution is a continuous process, how we stop the formation of drug resistant mutant alleles is a very concerning question. Usually, high mean parasitemia index is observed in *falciparum* infected persons but *vivax* infection generally exhibits low parasitemia index secondary to its preference to invade reticulocytes rather than erythrocytes [89, 90].

Resistance to chloroquine in *falciparum* is due to point mutations in the gene encoding *pfcrt* (*P. falciparum* chloroquine resistance transporter) and *pfmdr* (*P. falciparum* multidrug resistance protein [P-glycoprotein transporter proteins]), resulting in reduced drug accumulation in the food vacuole [91]. Chloroquineresistant *vivax* was first reported from Papua New Guinea in 1989. High grade chloroquine-resistant *vivax* is prevalent in areas such as Indonesia and Oceania (considered as chloroquine resistance epicenters) [92]. It is more challenging to detect chloroquine resistance in *vivax* since parasitemia is generally low relative to *falciparum*. In addition, it is not easy to distinguish *vivax* recrudescence from relapses as a result of reactivation of dormant hepatic parasites in endemic settings. Moreover, there is no robust *in vitro* culture system for *vivax*, so confirmation with *in vitro* susceptibility testing is even more challenging for *vivax* than for *falciparum*. Although *pvcrt-o* (*P. vivax* chloroquine resistance transporter-o) is orthologous to *pfcrt*, there is no clear direct association between chloroquine resistance and mutations in *pvcrt-o.* One current study in patients with recurrent *vivax* infections in the Brazilian Amazon found that chloroquine resistance was associated with increased copies of gene encoding *pvcrt-o* [88].

**Amodiaquine** and its slowly eliminated active metabolite (desethylamodiaquine) are structurally related to chloroquine, this explains the cross resistance observed in the field, where parasites were reported to harbor mutations on *pfcrt* and *pfmdr1* after amodiaquine treatment failure [93]. Therefore, amodiaquine is used in combination therapy with SP for prophylaxis and artesunate for treatment. DHA-piperaquine (co-formulated tablet) has shown excellent efficacy (without apparent drug resistance) and safety in treating *falciparum* malaria. But now, resistance has been reported from Western Cambodia to be associated with a point mutation of *pfcrt* and amplification of *plasmepsin* 2 and 3 genes in *falciparum* parasites. The *plasmepsin* genes encode aspartic proteases that function as hemoglobinases in the parasitie's digestive vacuole. The mechanism of resistance is not clearly known; however hypothesized that increased hemoglobin digestion due to the amplification decreases the reactive heme species concentrations that piperaquine binds, thereby overcoming the inhibition of heme detoxification by piperaquine [88].

Documents written regarding **quinine** resistance are rare, but isolated cases have been reported from Thailand, North India, East Africa and South America [93]. Resistance mechanisms to quinine appear to be more complex. *In vitro* cross resistance between quinine, other aryl aminoalcohols, and 4-aminoquinolines is observed, suggesting that there may be a common genetic mechanism of resistance among those drugs. Mutations in *pfmdr1* and *pfcrt* have been found to confer decreased susceptibility of the parasite to quinine. Yet, they are not sufficient to bring resistance, implying that there are additional genes involved. A quantitative trait loci analysis done to detect genes associated with quinine resistance in 71 *falciparum* isolates from diverse locations has been identified *pfmdr1*, *pfcrt*, and *pfnhe-1* (*P. falciparum Na*<sup>+</sup> */H*<sup>+</sup> *exchanger-1*). *Pfnhe-1* encodes *falciparum* Na+ /H+ exchanger 1 and is on chromosome 13. M**efloquine** resistance by both *falciparum* and *vivax* was found to be primarily mediated by *mdr1* amplification (increased *mdr1* copy number), rather than through point mutations similar to chloroquine and antifolate drugs. Resistance to **primaquine** in *vivax* is difficult to verify as it is confounded by reinfections in malaria-endemic regions. A research that done whole genome sequencing of *vivax* from known relapses that occurred despite primaquine therapy found polymorphisms in many putative resistance genes. However, there are currently no known genetic markers of primaquine and tafenoquine resistance [88].

In contrast to chloroquine resistance, which took many years to develop, antifolates resistance developed much faster. The genetic mechanism of resistance for antifolates is more straightforward than chloroquine resistance. The reason for resistanc against antifolates is single point-mutations in the genes encoding either DHFR—*pfdhfr* in *falciparum* and *pvdhfr* in *vivax* malaria, or DHPS—*pfdhps* in *falciparum and pvdhps* in *vivax* malaria. *Dhfr* mutations reduce the overall efficacy of the enzyme and result in a fitness cost for *Plasmodium*. Following changes in first-line therapy of malaria from sulfa-drugs to ACTs, a decline in triple and quadruple *dhfr* mutants has been observed in certain regions. Nonetheless, in nations where SP is part of the ACT or SP is used as intermittent preventive therapy (IPT), these mutants remain prevalent. In addition, the persistence of the *Plasmodium* species carrying *dhfr* mutations may be attributed to the use of trimethroprimsulfamethoxazole for prophylaxis or for treating opportunistic infections in HIV positive individuals. Interestingly, *falciparum* species in Southeast Asia are able to develop a compensatory mutation for the fitness cost incurred by the mutant *dhfr*. A genome scanning study of *falciparum* strains first identified an amplification surrounding GTP-cyclohydrolase 1 (*gch1*), which encodes an enzyme in the folate biosynthesis pathway that is upstream from DHFR and DHPS. The amplification reduces the cost of acquiring the drug-resistance mutations further downstream in the folate synthesis pathway [88]. **Atovaquone** acquires resistance related to a single mutation of cytochrome b gene of the parasite [68].

**Artemisinin** resistance in *falciparum* has currently been detected in five countries of Greater Mekong sub-region (Cambodia, Lao People's Democratic Republic, Myanmar, Thailand and Viet Nam). These resistant *Plasmodium* strains have the ability of spreading into many world countries including Africa and then they become a global threat for malaria control and treatment [13, 94]. Though different studies associate artemisinin resistance with mutuation in *pfatp6* (*P. falciparum encoded sarcoendoplasmic reticulum Ca*2+*-ATPase6*), *pfmdr1*, *pffd* (*P. falciparum ferredoxin*), *pfarps10* (*apicoplast ribosomal protein s10*), *pfmdr2* or *pfcrt* genes of falciparum, these mutations are thought to represent a background upon which the *kelch13* mutations are especially likely to occur. The genetic mediator(s) of *vivax* resistance against artemisinins is/are not reported till now. Lumefantrine resistance in field isolates has not yet been convincingly demonstrated. However, amplification of the *pfmdr1* gene in *falciparum* and *pvmdr1* in *vivax* has been associated with increased risk of treatment failure of coartem®. Antibacterials such as tetracycline, doxycycline, clindamycin and azithromycin also have antiplasmodial activity although in general their action is slow for malaria treatment. They are recommended only in combination with other antimalarials. Apicoplast ribosomal RNA (23S rRNA) mutation mediated *falciparum* resistance to clindamycin has been found in field isolates. There are no clear markers of doxycycline resistance that have been identified thus far [88, 93].


**A.2 The probability density function of the student-t distribution is:**

$$f(\mathbf{y}, \boldsymbol{\nu}) = \frac{\Gamma^{\frac{\nu+1}{2}}}{\sqrt{\pi(\boldsymbol{\nu} - \mathbf{2})} \Gamma^{\frac{\nu}{2}} \left(\mathbf{1} + \frac{\boldsymbol{\nu}^2}{\boldsymbol{\nu} - \mathbf{2}}\right)^{\frac{\nu+1}{2}}} \tag{10}$$

Its log likelihood function in GARCH term is:

$$\log\left[\Gamma\left(\frac{\nu+1}{2}\right)\right] - \log\left[\Gamma\left(\frac{\nu}{2}\right)\right] - \frac{1}{2}\log\left(\pi(\nu-2)\right) - \frac{1}{2}\sum\_{j=1}^{n}\left[\log\left(h\_{l}\right) + (\nu+1)\log\left(1 + \frac{e\_{l}^{2}}{h\_{l}(\nu-2)}\right)\right] \tag{11}$$