*Plasmodium vivax* and *Plasmodium ovale* in the Malaria Elimination Agenda in Africa

*Isaac K. Quaye and Larysa Aleksenko*

#### **Abstract**

In recent times, several countries in sub-Saharan Africa have reported cases of *Plasmodium vivax (Pv)* with a considerable number being Duffy negative. Current efforts at malaria elimination are focused solely on *Plasmodium falciparum* (*Pf*) excluding non-falciparum malaria. *Pv* and *Plasmodium ovale (Po)* have hypnozoite forms that can serve as reservoirs of infection and sustain transmission. The burden of these parasites in Africa seems to be more than acknowledged, playing roles in migrant and autochthonous infections. Considering that elimination and eradication is a current aim for WHO and Roll Back Malaria (RBM), the inclusion of *Pv* and *Po* in the elimination agenda cannot be over-emphasized. The biology of *Pv* and *Po* are such that the same elimination strategies as are used for *Pf* cannot be applied so, going forward, new approaches will be required to attain elimination and eradication targets.

**Keywords:** *Plasmodium vivax* transmission, *Plasmodium ovale*, asymptomatic transmission, sub-Saharan Africa

#### **1. Introduction**

Malaria elimination is defined as interruption of local transmission of malaria in a defined area [1, 2]. Within a geographical demarcation, malaria transmission is heterogenous ranging between high, intermediate or low [3, 4]. This means that elimination efforts must be defined with respect to the geographical area, and interventions targeted to specified strata to achieve elimination goals [5, 6]. Data for such targeted intervention can come from the use of sensitive and specific surveillance diagnostic tools that can discriminate between species, including asymptomatic infections. In Africa, current efforts of intervention by National Malaria Control Programs and recommendation by WHO and Roll Back Malaria (RBM) are focused mainly on *Plasmodium falciparum* (*Pf*) with little to no attention on non-falciparum species although the latter are established to be present in all of Africa [7, 8]. Clearly if elimination is envisaged based on the target of RBM, then inclusion of all human *Plasmodium* species in the elimination agenda is relevant. Non- falciparum *Plasmodial* species are more complicated in their transmission than *Pf* [9–11]. Especially, *Plasmodium vivax (Pv),* that is the most prevalent in the world and the two sympatric species of *Plasmodium ovale* (*Po*), *Plasmodium ovale curtisi* and *Plasmodium ovale wallikeri* both of which cause severe disease and have

hypnozoite stages in their transmission cycle as *Pv* [12–15]. The inclusion of all species should be well planned in such a way that it does not take away from the efforts being made to reduce *Pf* transmission but to go hand in hand. The transmission dynamics of these species must be well understood so that measures tailored to them are put in place. Here aspects of the biology of the parasites are elaborated so they are put in perspective with regards to elimination.

#### **2. Biology of** *Pv* **and** *Po*

*Plasmodium* species are obligate intracellular parasites that infect *Anopheles* mosquitoes in the sexual life cycle and humans in their asexual life cycle [16]. In humans, replication takes place in the liver hepatocytes, following inoculation by an infected *Anopheles* mosquito into the skin, and migration of the parasite through the capillaries into the blood stream and then to the liver parenchyma cells [17]. This pattern of infection is similar in all known human *Plasmodium* parasites which currently are *P. falciparum*, *P. vivax*, *P. ovale curtisi*, *P. ovale wallikeri*, *P. malariae* and *P. knowlesi* [18].

Outside of Africa, *Pv* is the predominant parasite, but even so the phenotypic characteristic from individuals infected with the parasite from different populations appear to differ. One of the most important differences between plasmodial species infecting humans is the ability of *Pv* and *Po* to relapse after the cure of the original infection [9, 12]. A proportion of sporozoites do not undergo immediate development in the invaded hepatocytes. Instead, they remain dormant in the liver as hypnozoites for prolonged periods of time before developing and causing recurrent infection [19]. *P. vivax* strains from different geographical areas show widely different relapse patterns, reflecting evolutionary adaptation to local environmental conditions that optimize transmission potential of the parasite [20]. In Africa, it had previously been generally accepted that because of the high prevalence of ACKR1 polymorphism, individuals of African descent are resistant to infection by the parasite [21]. Recent evidence of *Pv* presence in nearly the whole of Africa, indicates that *Pv* may have other mechanisms for invasion [22]. Advancing research into the parasites' biology and mechanism of invasion is necessary for elimination and eradication. Although *Po* is not limited by the Duffy antigen polymorphism, the shared biology of hypnozoites and their relapse makes it of equal concern in malaria elimination. What is palpably clear is that reporting of these two parasites from countries in Africa, appears not to reflect the true prevalence rate as reporting is largely passive and not active.

#### **2.1 Parasite transmission**

Transmission of the parasite defines the processes that the parasite takes to complete a life cycle [1]. The process in the *Anopheles* vector is referred to as sporogony while that in humans is referred to as schizogony. Schizogony begins when liver schizonts which contain multiples of merozoites mature and burst with subsequent release of the merozoites into the blood stream [23]. The merozoites invade young reticulocytes that have the surface marker CD71(CD71+ ) [24] where they mature into trophozoites. The trophozoites mature into blood stage schizonts containing multiples of merozoites, burst the reticulocytes and initiate a new cycle of blood stage transmission. Some of the merozoites mature into male and female gametocytes that are picked by female *Anopheles* mosquitoes when mature, to begin the sporogonic cycle [1]. In *Pv* and *Po* some of the liver schizonts remain dormant for weeks, months or years and then get activated to initiate a new infection in the

**189**

Plasmodium vivax *and* Plasmodium ovale *in the Malaria Elimination Agenda in Africa*

reticulocytes [16]. It is not clear what exactly triggers the reactivation, although new *Plasmodium* and other infections and inflammation, have been suggested as contributory [9, 25]. The subsets that remain in the liver are the hypnozoites, and the reactivation process is called 'relapse'. The activation of hypnozoites is reported to cause most of the blood stage infections [26]. It is not clear whether the underreporting of *Pv* and *Po* infections in Africa is attributed to hypnozoites relapsing or

It has been reported that the hypnozoites are not only derived from liver parenchyma cells, but also from bone marrow parenchyma that abounds in young and

 reticulocytes [24]. It has been shown that the bone marrow is enriched in gametocytes extravascularly, while the liver is enriched with tissue schizonts in the sinusoids compared to peripheral blood [24]. In addition, a small number of parasites are seen in the vasculature of the lungs as opposed to those in the bone marrow and liver which are extravascular [27]. The hypnozoite stages are particularly troubling because chemotherapy against them with the use of primaquine is restrictive due to glucose-6-phosphate dehydrogenase deficiency and CYP2D6 polymorphism [28]. Also, it has been reported that primaquine may not be efficacious against some strains of the hypnozoite stages [29]. Nevertheless, radical cure with primaquine at

Previously there was no direct evidence of hypnozoite stages of *Po*, however recent reports have clearly shown that such stages indeed exist for both *Po curtisi* and *Po wallikeri* [30]. The sub-species of the parasite contribute to the transmission dynamics and need to be fully interrogated for markers and drug targeting. Another unique biology of the parasite is the early emergence and maturation of gametocytes in the blood. While gametocytes for *Pf*, *Pm* mature in about 10–12 days, for *Pv and Po*, gametocytes are seen between 3 to 5 days following the first documentation of parasites in the peripheral blood [1, 16, 23]. This means even before any symptoms of infection are seen the parasite would have been transmitted if female Anopheles mosquitoes fed within the period. In this case an intervention in the transmission process can be missed. It maybe that these traditionally unseen parasites, contribute to the low parasitemia usually seen with these infections and why *Pv* and *Po* infections are characterized as benign although they can cause as much severe disease as in *Pf* infections.

In regions outside of sub-Saharan Africa (SSA) where *Pf* and *Pv* coexist, mixed-species infections are common [31]. In such situations, there are observed shifts in the dominant parasite towards *Pv* as *Pf* transmission declines. Surveys usually report rates less than 2% and yet careful clinical studies record rates of up to 30% and this figure is even higher when PCR detection methods are used. This trend means that in a couple of years to come, without effective and necessary interventions, when the *Pf* burden has been significantly reduced in Africa, *Pv* and *Po* infections could constitute the most dominant *Plasmodium* species on the continent. We observe that countries that were on course for *Pf* elimination such as Botswana, eSwatini, and South Africa have seen changes recently, part of which is due to *Pv* and *Po*. Concurrent infections with different *Plasmodium* species may have important implications on the host response and development of cross-species immunity. The potential for *Pv* to attenuate *Falciparum* malaria obviously requires further characterization and has significant implications for vivax-only vaccination strategies, and the deployment of drugs such as chloroquine, which has lost efficacy

lack of tools or focus for targeting these species for detection.

least can go a long way in the agenda towards elimination.

*DOI: http://dx.doi.org/10.5772/intechopen.96867*

CD71+

**2.2 Hypnozoite stages**

**2.3 Transmission dynamics**

Plasmodium vivax *and* Plasmodium ovale *in the Malaria Elimination Agenda in Africa DOI: http://dx.doi.org/10.5772/intechopen.96867*

reticulocytes [16]. It is not clear what exactly triggers the reactivation, although new *Plasmodium* and other infections and inflammation, have been suggested as contributory [9, 25]. The subsets that remain in the liver are the hypnozoites, and the reactivation process is called 'relapse'. The activation of hypnozoites is reported to cause most of the blood stage infections [26]. It is not clear whether the underreporting of *Pv* and *Po* infections in Africa is attributed to hypnozoites relapsing or lack of tools or focus for targeting these species for detection.

It has been reported that the hypnozoites are not only derived from liver parenchyma cells, but also from bone marrow parenchyma that abounds in young and CD71+ reticulocytes [24]. It has been shown that the bone marrow is enriched in gametocytes extravascularly, while the liver is enriched with tissue schizonts in the sinusoids compared to peripheral blood [24]. In addition, a small number of parasites are seen in the vasculature of the lungs as opposed to those in the bone marrow and liver which are extravascular [27]. The hypnozoite stages are particularly troubling because chemotherapy against them with the use of primaquine is restrictive due to glucose-6-phosphate dehydrogenase deficiency and CYP2D6 polymorphism [28]. Also, it has been reported that primaquine may not be efficacious against some strains of the hypnozoite stages [29]. Nevertheless, radical cure with primaquine at least can go a long way in the agenda towards elimination.

#### **2.2 Hypnozoite stages**

*Current Topics and Emerging Issues in Malaria Elimination*

they are put in perspective with regards to elimination.

**2. Biology of** *Pv* **and** *Po*

and *P. knowlesi* [18].

is largely passive and not active.

**2.1 Parasite transmission**

hypnozoite stages in their transmission cycle as *Pv* [12–15]. The inclusion of all species should be well planned in such a way that it does not take away from the efforts being made to reduce *Pf* transmission but to go hand in hand. The transmission dynamics of these species must be well understood so that measures tailored to them are put in place. Here aspects of the biology of the parasites are elaborated so

*Plasmodium* species are obligate intracellular parasites that infect *Anopheles* mosquitoes in the sexual life cycle and humans in their asexual life cycle [16]. In humans, replication takes place in the liver hepatocytes, following inoculation by an infected *Anopheles* mosquito into the skin, and migration of the parasite through the capillaries into the blood stream and then to the liver parenchyma cells [17]. This pattern of infection is similar in all known human *Plasmodium* parasites which currently are *P. falciparum*, *P. vivax*, *P. ovale curtisi*, *P. ovale wallikeri*, *P. malariae*

Outside of Africa, *Pv* is the predominant parasite, but even so the phenotypic characteristic from individuals infected with the parasite from different populations appear to differ. One of the most important differences between plasmodial species infecting humans is the ability of *Pv* and *Po* to relapse after the cure of the original infection [9, 12]. A proportion of sporozoites do not undergo immediate development in the invaded hepatocytes. Instead, they remain dormant in the liver as hypnozoites for prolonged periods of time before developing and causing recurrent infection [19]. *P. vivax* strains from different geographical areas show widely different relapse patterns, reflecting evolutionary adaptation to local environmental conditions that optimize transmission potential of the parasite [20]. In Africa, it had previously been generally accepted that because of the high prevalence of ACKR1 polymorphism, individuals of African descent are resistant to infection by the parasite [21]. Recent evidence of *Pv* presence in nearly the whole of Africa, indicates that *Pv* may have other mechanisms for invasion [22]. Advancing research into the parasites' biology and mechanism of invasion is necessary for elimination and eradication. Although *Po* is not limited by the Duffy antigen polymorphism, the shared biology of hypnozoites and their relapse makes it of equal concern in malaria elimination. What is palpably clear is that reporting of these two parasites from countries in Africa, appears not to reflect the true prevalence rate as reporting

Transmission of the parasite defines the processes that the parasite takes to complete a life cycle [1]. The process in the *Anopheles* vector is referred to as sporogony while that in humans is referred to as schizogony. Schizogony begins when liver schizonts which contain multiples of merozoites mature and burst with subsequent release of the merozoites into the blood stream [23]. The merozoites

they mature into trophozoites. The trophozoites mature into blood stage schizonts containing multiples of merozoites, burst the reticulocytes and initiate a new cycle of blood stage transmission. Some of the merozoites mature into male and female gametocytes that are picked by female *Anopheles* mosquitoes when mature, to begin the sporogonic cycle [1]. In *Pv* and *Po* some of the liver schizonts remain dormant for weeks, months or years and then get activated to initiate a new infection in the

) [24] where

invade young reticulocytes that have the surface marker CD71(CD71+

**188**

Previously there was no direct evidence of hypnozoite stages of *Po*, however recent reports have clearly shown that such stages indeed exist for both *Po curtisi* and *Po wallikeri* [30]. The sub-species of the parasite contribute to the transmission dynamics and need to be fully interrogated for markers and drug targeting. Another unique biology of the parasite is the early emergence and maturation of gametocytes in the blood. While gametocytes for *Pf*, *Pm* mature in about 10–12 days, for *Pv and Po*, gametocytes are seen between 3 to 5 days following the first documentation of parasites in the peripheral blood [1, 16, 23]. This means even before any symptoms of infection are seen the parasite would have been transmitted if female Anopheles mosquitoes fed within the period. In this case an intervention in the transmission process can be missed. It maybe that these traditionally unseen parasites, contribute to the low parasitemia usually seen with these infections and why *Pv* and *Po* infections are characterized as benign although they can cause as much severe disease as in *Pf* infections.

#### **2.3 Transmission dynamics**

In regions outside of sub-Saharan Africa (SSA) where *Pf* and *Pv* coexist, mixed-species infections are common [31]. In such situations, there are observed shifts in the dominant parasite towards *Pv* as *Pf* transmission declines. Surveys usually report rates less than 2% and yet careful clinical studies record rates of up to 30% and this figure is even higher when PCR detection methods are used. This trend means that in a couple of years to come, without effective and necessary interventions, when the *Pf* burden has been significantly reduced in Africa, *Pv* and *Po* infections could constitute the most dominant *Plasmodium* species on the continent. We observe that countries that were on course for *Pf* elimination such as Botswana, eSwatini, and South Africa have seen changes recently, part of which is due to *Pv* and *Po*. Concurrent infections with different *Plasmodium* species may have important implications on the host response and development of cross-species immunity. The potential for *Pv* to attenuate *Falciparum* malaria obviously requires further characterization and has significant implications for vivax-only vaccination strategies, and the deployment of drugs such as chloroquine, which has lost efficacy against *Pf* but still retain it against *Pv.* The transmission of the parasite in Duffy negative individuals raises serious concern as essentially *Pf* is the only targeted species in the elimination agenda in Africa, with minimal consideration for *Pv and Po.*

#### **2.4 What is needed currently on** *Pv* **and** *Po* **transmission?**

As noted previously, *Pv* and *Po* can cause severe disease [13, 15]. The most characteristic of *Pv* are acute respiratory distress syndrome (ARDS), anemia and decreased oxygen saturation in both children and adults [32, 33]; clinical conditions which are also seen in *Po* infections [34–36]. The respiratory illnesses are associated with high mortality, with a higher risk in women. In the cited *Po* cases of ARDS, the patients were also HIV positive, so it is not clear if HIV facilitated development to full blown ARDS or not [32, 37]. The new tissue sequestration sites of *Pv* and *Po* mention previously, which are out of routine diagnostic procedures, means the task of eliminating *Pv* and *Po* is clearly not an easy one. When the unique biology of *Pv* and *Po* vis-à-vis the evidence of their presence in Africa are considered, there is a need for a paradigm shift regarding *Pv* and *Po* research by putting in place the following:


#### **3. Conclusion and future perspectives**

The presence of *Pv* and *Po* in Africa is certain. Malaria control programs generally focus on providing good vector control, early diagnosis, and access to effective antimalarial regimens, preferably with anti-gametocyte activity to reduce transmission. All these tools cannot be optimally employed without a knowledge of the transmission dynamics of all parasites within a community or country. Re-engaging the focus of NMCPs on non-falciparum malaria that harbor hypnozoites and that potentially could be a significant problem in the very near future is a necessity. Pooling of resources regionally and internationally are key elements for the fight against reducing the burden of non-falciparum malaria and their elimination. It is important that NMCPs and researchers from Universities and Research Institutions engage in crosstalk to facilitate accurate detection and surveillance, and generate the human resource required for sustaining these efforts.

**191**

**Author details**

Isaac K. Quaye1

and Technology, Accra, Ghana

\* and Larysa Aleksenko2

Faculty of Medicine, Lund University, Lund, Sweden

provided the original work is properly cited.

\*Address all correspondence to: isaac.quaye@regent.edu.gh

1 Department of Applied Health Sciences, Regent University College of Science

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

2 Division of Obstetrics and Gynecology, Department of Clinical Sciences,

Plasmodium vivax *and* Plasmodium ovale *in the Malaria Elimination Agenda in Africa*

The authors acknowledge the patience and support of the Intech Open team and

*DOI: http://dx.doi.org/10.5772/intechopen.96867*

The authors declare no conflict of interest.

**Acknowledgements**

**Conflict of interest**

our entire family in the write up.

Plasmodium vivax *and* Plasmodium ovale *in the Malaria Elimination Agenda in Africa DOI: http://dx.doi.org/10.5772/intechopen.96867*

#### **Acknowledgements**

*Current Topics and Emerging Issues in Malaria Elimination*

**2.4 What is needed currently on** *Pv* **and** *Po* **transmission?**

shift regarding *Pv* and *Po* research by putting in place the following:

*Pv* and *Po* infections

malaria research.

elimination activities.

agenda in sub-Saharan Africa.

**3. Conclusion and future perspectives**

the human resource required for sustaining these efforts.

against *Pf* but still retain it against *Pv.* The transmission of the parasite in Duffy negative individuals raises serious concern as essentially *Pf* is the only targeted species in the elimination agenda in Africa, with minimal consideration for *Pv and Po.*

As noted previously, *Pv* and *Po* can cause severe disease [13, 15]. The most characteristic of *Pv* are acute respiratory distress syndrome (ARDS), anemia and decreased oxygen saturation in both children and adults [32, 33]; clinical conditions which are also seen in *Po* infections [34–36]. The respiratory illnesses are associated with high mortality, with a higher risk in women. In the cited *Po* cases of ARDS, the patients were also HIV positive, so it is not clear if HIV facilitated development to full blown ARDS or not [32, 37]. The new tissue sequestration sites of *Pv* and *Po* mention previously, which are out of routine diagnostic procedures, means the task of eliminating *Pv* and *Po* is clearly not an easy one. When the unique biology of *Pv* and *Po* vis-à-vis the evidence of their presence in Africa are considered, there is a need for a paradigm

1.Sensitization of National Malaria Control Programs (NMCPs) to the menace

2.Provision of baseline and standardized tools for sample collection, detection

3.Measures for all individuals irrespective of age in Africa to be at reduced risk to

4.De-escalation of the burden of *Pv and Po,* through the utilization of novel and available tools that can be integrated into malaria control and elimination

5.Crosstalk between NMCPs and researchers as well as institutions engaged in

6.Creation of fora for exchange of knowledge and resources between *Pv* and *Po* focused scientists in Africa and globally, to facilitate progress in control and

The presence of *Pv* and *Po* in Africa is certain. Malaria control programs generally focus on providing good vector control, early diagnosis, and access to effective antimalarial regimens, preferably with anti-gametocyte activity to reduce transmission. All these tools cannot be optimally employed without a knowledge of the transmission dynamics of all parasites within a community or country. Re-engaging the focus of NMCPs on non-falciparum malaria that harbor hypnozoites and that potentially could be a significant problem in the very near future is a necessity. Pooling of resources regionally and internationally are key elements for the fight against reducing the burden of non-falciparum malaria and their elimination. It is important that NMCPs and researchers from Universities and Research Institutions engage in crosstalk to facilitate accurate detection and surveillance, and generate

of the two parasites in asymptomatic and symptomatic infections

and assays for *Pv* and *Po* towards the elimination of the parasites.

**190**

The authors acknowledge the patience and support of the Intech Open team and our entire family in the write up.

## **Conflict of interest**

The authors declare no conflict of interest.

## **Author details**

Isaac K. Quaye1 \* and Larysa Aleksenko2

1 Department of Applied Health Sciences, Regent University College of Science and Technology, Accra, Ghana

2 Division of Obstetrics and Gynecology, Department of Clinical Sciences, Faculty of Medicine, Lund University, Lund, Sweden

\*Address all correspondence to: isaac.quaye@regent.edu.gh

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[21] L.H. Miller, S.J. Mason, D.F. Clyde, M.H. McGinniss, The resistance factor to *Plasmodium vivax* in blacks. The Duffy-blood-group genotype, FyFy, N. Engl. J. Med. 295 (1976) 302-304.

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Plasmodium vivax *and* Plasmodium ovale *in the Malaria Elimination Agenda in Africa DOI: http://dx.doi.org/10.5772/intechopen.96867*

Severe Plasmodium ovale malaria complicated by acute respiratory distress syndrome in a young Caucasian man, Malar J. 17 (2018) 139-018-2289-2.

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[20] L.J. Robinson, R. Wampfler, I. Betuela, S. Karl, M.T. White, C.S. Li Wai Suen, N.E. Hofmann, B. Kinboro, A. Waltmann, J. Brewster, L. Lorry, N. Tarongka, L. Samol, M. Silkey, Q. Bassat, P.M. Siba, L. Schofield, I. Felger, I. Mueller, Strategies for understanding and reducing the *Plasmodium vivax* and Plasmodium ovale hypnozoite reservoir in Papua New Guinean children: a randomised placebo-controlled trial and mathematical model, PLoS Med. 12 (2015) e1001891.

[21] L.H. Miller, S.J. Mason, D.F. Clyde, M.H. McGinniss, The resistance factor to *Plasmodium vivax* in blacks. The Duffy-blood-group genotype, FyFy, N. Engl. J. Med. 295 (1976) 302-304.

[22] P.A. Zimmerman, Plasmodium vivax Infection in Duffy-Negative People in Africa, Am. J. Trop. Med. Hyg. 97 (2017) 636-638.

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[28] J.K. Baird, K.E. Battle, R.E. Howes, Primaquine ineligibility in anti-relapse therapy of *Plasmodium vivax* malaria: the problem of G6PD deficiency and cytochrome P-450 2D6 polymorphisms, Malar J. 17 (2018) 42-018-2190-z.

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[13] S. Amireh, H. Shaaban, G. Guron, Severe Plasmodium vivax cerebral malaria complicated by hemophagocytic

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[5] T. Bousema, G. Stresman, A.Y. Baidjoe, J. Bradley, P. Knight, W. Stone, V. Osoti, E. Makori, C. Owaga, W. Odongo, P. China, S. Shagari, O.K. Doumbo, R.W. Sauerwein, S. Kariuki, C. Drakeley, J. Stevenson, J. Cox, The Impact of Hotspot-Targeted Interventions on Malaria Transmission in Rachuonyo South District in the Western Kenyan Highlands: A Cluster-Randomized Controlled Trial, PLoS

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[31] P.A. Zimmerman, R.K. Mehlotra, L.J. Kasehagen, J.W. Kazura, Why do we need to know more about mixed Plasmodium species infections in humans? Trends Parasitol. 20 (2004) 440-447.

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[33] N.M. Anstey, B. Russell, T.W. Yeo, R.N. Price, The pathophysiology of vivax malaria, Trends Parasitol. 25 (2009) 220-227.

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[35] Y.L. Lau, W.C. Lee, L.H. Tan, A. Kamarulzaman, S.F. Syed Omar, M.Y. Fong, F.W. Cheong, R. Mahmud, Acute respiratory distress syndrome and acute renal failure from Plasmodium ovale infection with fatal outcome, Malar J. 12 (2013) 389-2875-12-389.

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**Chapter 12**

*and Eugenia Lo*

**Abstract**

**1. Introduction**

**195**

a severe form and result in death [6].

Progress in Parasite Genomics and

A wide deployment of malaria control tools have significantly reduced malaria morbidity and mortality across Africa. However, in the last five to seven years, there has been a resurgence of malaria in several African countries, raising the questions of whether and why current control mechanisms are failing. Since the first *Plasmodium falciparum* reference genome was published in 2002, few thousands more representing a broad range of geographical isolates have been

sequenced. These advances in parasite genomics have improved our understanding of mutational changes, molecular structure, and genetic mechanisms associated with diagnostic testing, antimalarial resistance, and preventive measures such as vaccine development. In this chapter, we summarize the current progress on: (1) genomic characteristics of *P. falciparum*; (2) novel biomarkers and revolutionary techniques for diagnosing malaria infections; and (3) current vaccine targets and

**Keywords:** genomics, *Plasmodium falciparum*, malaria diagnosis, bioinformatics,

Malaria remains a serious public health problem in several developing countries. Globally, there are about 3.2 billion people at risk of malaria and 435,000 malariarelated deaths, most of which happening in West Africa [1, 2]. Malaria is a compli-

Apicomplexa. While *P. falciparum* is the most prevalent form of malaria, *P. vivax* is most widespread around the world. *Plasmodium* has a complicated life cycle that reproduces asexually in human and sexually in mosquito hosts. The parasite is transmitted by the female *Anopheles* mosquitoes. *Anopheles* mosquitoes thrive in warm, tropical climates as the temperature allows for quicker breeding and hatching [3]. The abundance of vectors is positively correlated with the transmission rate of malaria [4]. People infected with malaria normally experience fever, chills, diarrhea, vomiting, and anemia [5]. If remain untreated, the disease can progress to

challenges for developing efficacious and long-lasting malaria vaccines.

antimalarial resistance, malaria vaccines, biomarkers, genetic diversity

cated disease caused by the genus *Plasmodium* in the protozoan phylum

Its Application to Current

Challenges in Malaria Control

*Cheikh Cambel Dieng, Colby T. Ford, Jennifer Huynh,*

*Linda E. Amoah, Yaw A. Afrane, Daniel A. Janies*

#### **Chapter 12**

*Current Topics and Emerging Issues in Malaria Elimination*

[37] M.V. Lacerda, S.C. Fragoso, M.G. Alecrim, M.A. Alexandre, B.M.

Becerra, H. del Portillo, J. Ordi, P.L. Alonso, Q. Bassat, Postmortem characterization of patients with clinical diagnosis of Plasmodium vivax malaria: to what extent does this parasite kill? Clin. Infect. Dis. 55 (2012) e67–e74.

Magalhaes, A.M. Siqueira, L.C. Ferreira, J.R. Araujo, M.P. Mourao, M. Ferrer, P. Castillo, L. Martin-Jaular, C. Fernandez-

medicines in radical cure of Plasmodium vivax, Acta Trop. 160 (2016) 35-38.

Mischlinger, R. Manego Zoleko, J. Kim, A. Klicpera, E.L. Meyer, D. Blessborn, M. Winterberg, A.A. Adegnika, S.T. Agnandji, P.G. Kremsner, B. Mordmuller, G. Mombo-Ngoma, H.P. Fuehrer, M. Ramharter, Prospective Clinical and Molecular Evaluation of Potential Plasmodium ovale curtisi and wallikeri Relapses in a Hightransmission Setting, Clin. Infect. Dis.

[31] P.A. Zimmerman, R.K. Mehlotra, L.J. Kasehagen, J.W. Kazura, Why do we need to know more about mixed Plasmodium species infections in humans? Trends Parasitol. 20 (2004)

[32] E. Tjitra, N.M. Anstey, P. Sugiarto, N. Warikar, E. Kenangalem, M. Karyana, D.A. Lampah, R.N. Price, Multidrug-resistant Plasmodium vivax associated with severe and fatal malaria: a prospective study in Papua, Indonesia,

[33] N.M. Anstey, B. Russell, T.W. Yeo, R.N. Price, The pathophysiology of vivax malaria, Trends Parasitol. 25

[34] K.K. Dayananda, R.N. Achur, D.C. Gowda, Epidemiology, drug resistance, and pathophysiology of Plasmodium vivax malaria, J. Vector Borne Dis. 55

[35] Y.L. Lau, W.C. Lee, L.H. Tan, A. Kamarulzaman, S.F. Syed Omar, M.Y. Fong, F.W. Cheong, R. Mahmud, Acute respiratory distress syndrome and acute renal failure from Plasmodium ovale infection with fatal outcome, Malar J. 12

[36] S. Eiam-Ong, Malarial nephropathy,

Semin. Nephrol. 23 (2003) 21-33.

(2013) 389-2875-12-389.

PLoS Med. 5 (2008) e128.

(2009) 220-227.

(2018) 1-8.

[30] M. Groger, L. Veletzky, A. Lalremruata, C. Cattaneo, J.

69 (2019) 2119-2126.

440-447.

**194**

## Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control

*Cheikh Cambel Dieng, Colby T. Ford, Jennifer Huynh, Linda E. Amoah, Yaw A. Afrane, Daniel A. Janies and Eugenia Lo*

#### **Abstract**

A wide deployment of malaria control tools have significantly reduced malaria morbidity and mortality across Africa. However, in the last five to seven years, there has been a resurgence of malaria in several African countries, raising the questions of whether and why current control mechanisms are failing. Since the first *Plasmodium falciparum* reference genome was published in 2002, few thousands more representing a broad range of geographical isolates have been sequenced. These advances in parasite genomics have improved our understanding of mutational changes, molecular structure, and genetic mechanisms associated with diagnostic testing, antimalarial resistance, and preventive measures such as vaccine development. In this chapter, we summarize the current progress on: (1) genomic characteristics of *P. falciparum*; (2) novel biomarkers and revolutionary techniques for diagnosing malaria infections; and (3) current vaccine targets and challenges for developing efficacious and long-lasting malaria vaccines.

**Keywords:** genomics, *Plasmodium falciparum*, malaria diagnosis, bioinformatics, antimalarial resistance, malaria vaccines, biomarkers, genetic diversity

#### **1. Introduction**

Malaria remains a serious public health problem in several developing countries. Globally, there are about 3.2 billion people at risk of malaria and 435,000 malariarelated deaths, most of which happening in West Africa [1, 2]. Malaria is a complicated disease caused by the genus *Plasmodium* in the protozoan phylum Apicomplexa. While *P. falciparum* is the most prevalent form of malaria, *P. vivax* is most widespread around the world. *Plasmodium* has a complicated life cycle that reproduces asexually in human and sexually in mosquito hosts. The parasite is transmitted by the female *Anopheles* mosquitoes. *Anopheles* mosquitoes thrive in warm, tropical climates as the temperature allows for quicker breeding and hatching [3]. The abundance of vectors is positively correlated with the transmission rate of malaria [4]. People infected with malaria normally experience fever, chills, diarrhea, vomiting, and anemia [5]. If remain untreated, the disease can progress to a severe form and result in death [6].

A wide deployment of malaria control tools in the past few decades have significantly reduced malaria morbidity and mortality worldwide. The number of countries with fewer than 100 clinical malaria cases increased from six to 27. Countries including Iran, Malaysia, Timor-Leste Belize, Cabo Verde, China, and El Salvador reported zero malaria cases in 2019 and malaria cases dropped by 90% in the Greater Mekong subregion (GMS) [7]. However, in the last five to seven years, there has been a resurgence of malaria in several African countries, raising the questions of whether and why current control mechanisms are failing. A number of factors has limited malaria control and elimination efforts. First, *Plasmodium* isolates may respond differently to antimalarial drugs, with some evolved to become more resistant than the others due to prolonged drug use. Second, multiplicity of infection (MOI), i.e., the number of *Plasmodium* isolates co-infecting a single host, has made molecular characterization of the parasites and understanding of disease severity difficult [8]. Third, diagnostic inaccuracy related to false negative results by rapid diagnostic tests (RDTs) is becoming a more widespread phenomenon [9]. Other factors such as asymptomatic reservoirs leading to transmission, lack of effective vaccine, and warmer climates and changing environments caused by human activities [10] have also hampered malaria elimination efforts. Thus, it is critically important to create new tools that allow us to monitor parasite changes and use that information to improve existing control strategies. In this chapter, we will summarize the current progress on: (1) genomic characteristics of *P. falciparum*; (2) novel biomarkers and revolutionary techniques for diagnosing malaria infections; and (3) current vaccine targets and challenges for developing efficacious and long-lasting malaria vaccines.

transporters, but more genes for immune evasion to support host–parasite interactions [14]. With an average length of 2.4 Kb, *P. falciparum* genes are considerably larger than many organisms. *P. falciparum*'s genome contains a full set of transfer RNA (tRNA) ligase genes with minimal redundancy. 43 tRNAs have been identified to bind all codons except TGT and TGC, which code for cysteine, thus giving *P. falciparum* a slightly different amino acid translation than is seen in humans and other eukaryotes. By contrast, the mitochondrial genome of *P. falciparum* is only about 6Kb and does not contain any genes that encode for tRNAs, implying that the mitochondrion must import tRNAs from elsewhere into the cells [19, 20]. Polymorphic genes in the *P. falciparum* genome are useful in creating linkage maps to monitor mutational changes and genetic diversity of the parasites in response to malaria interventions and control efforts [21]. Polymorphic genes are variations in genes at higher frequencies that can be advantageous, neutral, or disadvantageous [22]. The role of polymorphic genes is to influence coding regions, alter protein sequences and gene expression, and eventually the metabolic pathway and function [23]. For example, remarkable polymorphisms observed in the merozoite surface proteins (*MSP*), *PfAMA1*, *PfEBA*, and *PfRHs* genes that involved in merozoite evasion have been shown to increase the evasion ability of *P. falciparum* to the host

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control*

*DOI: http://dx.doi.org/10.5772/intechopen.96530*

In comparison, conserved genes are genes that have not been altered. They contribute to important biological processes and fitness [25]. Information of conserved genes allow us to infer phylogenetic relatedness and trace the genetic origin of different lineages, determine new targets for therapeutic treatment, and serve as a guide when determining functions of unknown genes [18]. The processes that determine the polymorphisms of the parasitic genome include both selective pressures and recombination frequencies. Selective pressures on conserved genes allow certain important genes to remain unmodified for normal metabolic activities, whereas polymorphic genes diversify through frequent recombination allow for better evasion of antimalarial drug treatments and escape detection from diagnostic tests [26]. For example, the *PfHRP2* gene has shown with partial deletions and/or mutations over the past few years due to the usage of *HRP2*-specific Rapid Diagnostic tests (RDTs) [27, 28]. More specifically, the histidine-rich repeats in the *PfHRP2* gene drastically change the length of the gene and the ability of the parasites to evade RDTs. Therefore, novel genes that encode for parasite specific function might be potential new targets for malaria diagnosis and/or treatment [14]. It is important to distinguish metabolic pathways that the parasites use for invasion of

*P. falciparum* is clearly genetically distinct by geographical regions [29, 30]. Given the complex life cycle of *Plasmodium*, genomic data coupled with Genome-Wide Association Studies can offer deep insights into the tangled relationships among humans, mosquitoes, and the parasites. To date, databases such as

PlasmoDB have integrated sequence information, functional genomics, and annotation of data emerging from the *P. falciparum* genome sequencing consortium [31]. These databases provide an important platform to retrieve biological meaning from new 'omic' data and enhance diagnosis and treatment of infections caused by this

One of the challenges to malaria elimination is the burden of submicroscopic asymptomatic infections that contribute to malaria transmission [7]. The gold standard for malaria diagnosis is microscopy [32]. Microscopy can differentiate malaria

the host cells as well as evasion of the host immune system.

**3. Conventional and novel methods for malaria diagnosis**

immune system [24].

dreadful malaria pathogen.

**197**

#### **2. Genomic characteristics of** *Plasmodium falciparum*

Knowledge of the evolution and genetic variation of the *Plasmodium* genome offers incredible insights into novel means of malaria diagnosis and treatment. The advances in parasite genomics have improved our understanding of mutational changes, molecular structure, and genetic mechanisms associated with failure in diagnostic testing [11], antimalarial resistance [12], and preventive measures such as vaccine development [13]. Since the first reference genome of *P. falciparum* was published in 2002 [14], several thousand DNA sequences have been collected and deposited in public databases. *Plasmodium falciparum* genome is approximately 23.7 Mb with 14 chromosomes, a plasmid of about 35 kb, and lots of mitochondrial DNA copies of about 6 kb [15]. There are currently 5,438 genes that have been predicted/discovered within the genome with 33% uncertainty of their functions [16]. The genome contains many rich AT regions in both exons and introns (80% and 90%, respectively), which has some advantages and disadvantages when learning more about the genetic architecture of the parasites [17]. The advantage associated with the genomic data would be using its rich polymorphic AT content as biomarkers to map out the evolutionary structure of the parasites and correlating it with any drug resistant genes [15]. Although having a rich AT genome has contributed to a high yield in microsatellites or simple sequence repeats, there are some disadvantages when using the genome in genetic studies [15]. For example, within CRISPR-Cas9, the high AT content resulted in a decrease in the amount of gRNA target sites needed [18]. A deeper knowledge of the genome's polymorphic and conserved genes are therefore essential towards understanding the evolutionary timeline of various *P. falciparum* lineages.

Compared to other eukaryotic organisms, whole genome sequencing (WGS) showed that the genome of *P. falciparum* contains fewer genes for enzymes and

#### *Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control DOI: http://dx.doi.org/10.5772/intechopen.96530*

transporters, but more genes for immune evasion to support host–parasite interactions [14]. With an average length of 2.4 Kb, *P. falciparum* genes are considerably larger than many organisms. *P. falciparum*'s genome contains a full set of transfer RNA (tRNA) ligase genes with minimal redundancy. 43 tRNAs have been identified to bind all codons except TGT and TGC, which code for cysteine, thus giving *P. falciparum* a slightly different amino acid translation than is seen in humans and other eukaryotes. By contrast, the mitochondrial genome of *P. falciparum* is only about 6Kb and does not contain any genes that encode for tRNAs, implying that the mitochondrion must import tRNAs from elsewhere into the cells [19, 20]. Polymorphic genes in the *P. falciparum* genome are useful in creating linkage maps to monitor mutational changes and genetic diversity of the parasites in response to malaria interventions and control efforts [21]. Polymorphic genes are variations in genes at higher frequencies that can be advantageous, neutral, or disadvantageous [22]. The role of polymorphic genes is to influence coding regions, alter protein sequences and gene expression, and eventually the metabolic pathway and function [23]. For example, remarkable polymorphisms observed in the merozoite surface proteins (*MSP*), *PfAMA1*, *PfEBA*, and *PfRHs* genes that involved in merozoite evasion have been shown to increase the evasion ability of *P. falciparum* to the host immune system [24].

In comparison, conserved genes are genes that have not been altered. They contribute to important biological processes and fitness [25]. Information of conserved genes allow us to infer phylogenetic relatedness and trace the genetic origin of different lineages, determine new targets for therapeutic treatment, and serve as a guide when determining functions of unknown genes [18]. The processes that determine the polymorphisms of the parasitic genome include both selective pressures and recombination frequencies. Selective pressures on conserved genes allow certain important genes to remain unmodified for normal metabolic activities, whereas polymorphic genes diversify through frequent recombination allow for better evasion of antimalarial drug treatments and escape detection from diagnostic tests [26]. For example, the *PfHRP2* gene has shown with partial deletions and/or mutations over the past few years due to the usage of *HRP2*-specific Rapid Diagnostic tests (RDTs) [27, 28]. More specifically, the histidine-rich repeats in the *PfHRP2* gene drastically change the length of the gene and the ability of the parasites to evade RDTs. Therefore, novel genes that encode for parasite specific function might be potential new targets for malaria diagnosis and/or treatment [14]. It is important to distinguish metabolic pathways that the parasites use for invasion of the host cells as well as evasion of the host immune system.

*P. falciparum* is clearly genetically distinct by geographical regions [29, 30]. Given the complex life cycle of *Plasmodium*, genomic data coupled with Genome-Wide Association Studies can offer deep insights into the tangled relationships among humans, mosquitoes, and the parasites. To date, databases such as PlasmoDB have integrated sequence information, functional genomics, and annotation of data emerging from the *P. falciparum* genome sequencing consortium [31]. These databases provide an important platform to retrieve biological meaning from new 'omic' data and enhance diagnosis and treatment of infections caused by this dreadful malaria pathogen.

#### **3. Conventional and novel methods for malaria diagnosis**

One of the challenges to malaria elimination is the burden of submicroscopic asymptomatic infections that contribute to malaria transmission [7]. The gold standard for malaria diagnosis is microscopy [32]. Microscopy can differentiate malaria

A wide deployment of malaria control tools in the past few decades have significantly reduced malaria morbidity and mortality worldwide. The number of countries with fewer than 100 clinical malaria cases increased from six to 27. Countries including Iran, Malaysia, Timor-Leste Belize, Cabo Verde, China, and El Salvador reported zero malaria cases in 2019 and malaria cases dropped by 90% in the Greater Mekong subregion (GMS) [7]. However, in the last five to seven years, there has been a resurgence of malaria in several African countries, raising the questions of whether and why current control mechanisms are failing. A number of factors has limited malaria control and elimination efforts. First, *Plasmodium* isolates may respond differently to antimalarial drugs, with some evolved to become more resistant than the others due to prolonged drug use. Second, multiplicity of infection (MOI), i.e., the number of *Plasmodium* isolates co-infecting a single host, has made molecular characterization of the parasites and understanding of disease severity difficult [8]. Third, diagnostic inaccuracy related to false negative results by rapid diagnostic tests (RDTs) is becoming a more widespread phenomenon [9]. Other factors such as asymptomatic reservoirs leading to transmission, lack of effective vaccine, and warmer climates and changing environments caused by human activities [10] have also hampered malaria elimination efforts. Thus, it is critically important to create new tools that allow us to monitor parasite changes and use that information to improve existing control strategies. In this chapter, we

will summarize the current progress on: (1) genomic characteristics of *P. falciparum*; (2) novel biomarkers and revolutionary techniques for diagnosing malaria infections; and (3) current vaccine targets and challenges for developing

Knowledge of the evolution and genetic variation of the *Plasmodium* genome offers incredible insights into novel means of malaria diagnosis and treatment. The advances in parasite genomics have improved our understanding of mutational changes, molecular structure, and genetic mechanisms associated with failure in diagnostic testing [11], antimalarial resistance [12], and preventive measures such as vaccine development [13]. Since the first reference genome of *P. falciparum* was published in 2002 [14], several thousand DNA sequences have been collected and deposited in public databases. *Plasmodium falciparum* genome is approximately 23.7 Mb with 14 chromosomes, a plasmid of about 35 kb, and lots of mitochondrial DNA copies of about 6 kb [15]. There are currently 5,438 genes that have been predicted/discovered within the genome with 33% uncertainty of their functions [16]. The genome contains many rich AT regions in both exons and introns (80% and 90%, respectively), which has some advantages and disadvantages when learning more about the genetic architecture of the parasites [17]. The advantage associated with the genomic data would be using its rich polymorphic AT content as biomarkers to map out the evolutionary structure of the parasites and correlating it with any drug resistant genes [15]. Although having a rich AT genome has contributed to a high yield in microsatellites or simple sequence repeats, there are some disadvantages when using the genome in genetic studies [15]. For example, within CRISPR-Cas9, the high AT content resulted in a decrease in the amount of gRNA target sites needed [18]. A deeper knowledge of the genome's polymorphic and conserved genes are therefore essential towards understanding the evolutionary

Compared to other eukaryotic organisms, whole genome sequencing (WGS) showed that the genome of *P. falciparum* contains fewer genes for enzymes and

**2. Genomic characteristics of** *Plasmodium falciparum*

efficacious and long-lasting malaria vaccines.

*Current Topics and Emerging Issues in Malaria Elimination*

timeline of various *P. falciparum* lineages.

**196**

species and quantify parasite density, but only has a detection threshold of 10 to 50 parasites μl of blood (approximately 0.001% parasitemia, assuming an erythrocyte count of 5 106 cells μl) [33]. However, it requires well trained microscopists and is labor-intensive and time-consuming [32]. Previous studies have reported higher prevalence of asymptomatic parasitemia (6–7 times higher) using PCR compared to microscopy [5]. Nested PCR of the *18 s* rRNA genes has been commonly used to detect submicroscopic infections. PCR-based methods are also more sensitive than microscopy at detecting gametocytes particularly in asymptomatic individuals with low-density infections [34]. However, recent studies indicated a relatively high number of misdiagnosed infections [9], possibly due to low parasite density being less detectable by conventional PCR. The *18 s* rRNA gene has a reported detection thresholds of 0.5–5 parasites μl [35], but parasite density of asymptomatic infections especially in low transmission settings could be well below this threshold. As a result, many of these infections remain undetected in the general populations of several malaria-endemic countries [10, 36, 37] and provide perfect reservoirs for transmission at any time. Therefore, it is crucial to identify new gene targets or novel tools that are convenient and affordable for detecting asymptomatic infections, particularly in countries approaching elimination phase. Below are comparisons of various conventional and novel detection methods.

bonding affinity to proteins on a solid surface [50]. Samples, including and positive and negative controls are inserted on a 96 well plate which enables the possibility to analyze multiple samples simultaneously, then samples get incubated and detection of the signal is generated directly or indirectly via secondary tag on the specific antibody [51]. ELISA can be very efficient diagnostic tool for *Plasmodium* because it is highly specific and sensitive and therefore can be used to screen blood donors and pregnant women who may or may not have been exposed to the parasite [1]. However there are some limitations associated with using ELISA, as it is unable to detect antibodies in patients with acute infections, patients with different *Plasmodium* species, and various antigens within the different life stages of the parasite [52]. A recent study showed a lower detection threshold (3 parasites/μl) by ELISA test of the *Plasmodium* lactate dehydrogenase (*pLDH*) as compared to RDT (50–60 parasites/μl) [53]. However, ELISA has a lower sensitivity (69.9%) to antigens than RDT (88%) [53], and can be expensive and require a trained technician to operate

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control*

One of the current diagnostic tests in development is an antibody-based lateral flow immunoassay (LFIA). Unlike ELISA that requires repeated incubation and washing steps, LFIA is considered to be a simple, user-friendly and cost-effective method for front-line diagnosis [54]. LFIA is versatile enough to detect target genes in sample matrices including whole blood, saliva, and urine. This method is primarily applied to detecting gametocytes in malaria-infected samples. Gametocytes are the sexual form of the parasite that gets transmitted to the mosquito host. They play a very important role in malaria transmission, and can contribute to up to 80% [55] of the infectious reservoirs. Previous studies have shown that infections with parasitemia as low as 4 gametocytes/μl can sustain transmission [56]. LFIA targets *Pfs25* in infected blood samples, which is a glycophosphatidylinositol-linked protein expressed on the surface of *P. falciparum* zygotes and ookinetes but only found on female gametocytes in the human hosts [57]. The detection limit of *Pfs25* LFIA is 0.02 gametocytes/μl, much more sensitive than *pfhrp2* RDT with a detection limit of 50–100 parasites per μL of blood [35, 58]. Another protein at the surface of the female gametocyte *PSSP17*, which is presumably more abundant in saliva samples, was also investigated in Cameroon, Zambia and Sierra Leone with an estimated sensitivity of 83% (95% CI, 61 to 95) in symptomatic patients when compared to PCR as the gold standard [58]. Detection of *pfhrp2* in saliva have been reported recently using LFIA, but the sensitivity was only shown to be improved in severe to moderate form of infections with parasitemia >60,000 parasites/μL [59]. Although convenient, *pfhrp2*-based LFIA could be less

The combination of PCR-based assays and sequencing technologies have revolutionized malaria diagnosis since their introduction in the early 2000s. Various gene targets have be used to monitor genetic and/or mutational changes in the parasites that cause the disease. PCR is a very common technique used in malaria diagnoses in laboratory settings. The main advantage of this technique is that it enables us to identify individuals with low parasitemia (**Table 1**). There are multiple versions of this technique including nested conventional PCR, real-time quantitative PCR and reverse transcriptase PCR. Nested PCR is the easiest and least expensive methods among the others. It only requires a thermocycler, set of primers, reagents and visualization after gel electrophoresis. It is mostly a

and interpret the results [53].

**3.3 Lateral flow immunoassay (LFIA)**

*DOI: http://dx.doi.org/10.5772/intechopen.96530*

useful in detecting asymptomatic infections.

**3.4 PCR-based methods**

**199**

#### **3.1 Rapid diagnostic test (RDT)**

Rapid Diagnostic Test (RDT) is a lateral flow immunochromatographic test that can detect the presence of *Plasmodium* parasites by using blood from patients. The blood samples collected from patients must be lysed before the *Plasmodium* antigens being stained. RDT will indicate if the patient has malaria or not [38]. This diagnostic method is useful in rural areas as it is inexpensive and does not require expertise to perform the test [38]. There are various types of RDTs that can detect different antigens of *Plasmodium* parasites including *P. falciparum* and *P. vivax*, namely *P. falciparum* histidine-rich protein 2 (*PfHRP2*) and lactate dehydrogenase (*PfLDH*). However, recent studies indicated that parasites lacking the *pfhrp2*/*pfhrp3* genes could result in false-negative *PfHRP2*-RDT results, and these *pfhrp2*/*pfhrp3* variants have been spreading in many East and West African countries [39–42]. Reports of deletion of *HRP2* and *HRP3* genes was first reported in 2010 within the Peruvian Amazon [43] and the number of false negative RDTs has substantially increased in late 2014 [44]. Prevalence of false negative cases related to *HRP2* deletions in South America (with the exception of Peru) is lower than Asia and Sub-Saharan Africa [45]. The highest prevalence (50% *pfhrp2* deletions among all positive cases) were reported in Cambodia, Peru, and Eritrea [46]. Such a high prevalence could be explained by technical errors, mutations, selection and spread of the *pfhrp2* and 3 variants [47] from South America to Africa and Asia [41]. Recently, a novel HRP2 based Alere™ Malaria Ag P.f RDT (uRDT) was developed specifically for the detection of asymptomatic infections. Compared to the existing RDTs, the uRDT showed a greater than 10-fold improvement in diagnosing clinical and asymptomatic cases [48]. However, the sensitivities of the uRDT were inconsistent among different transmission settings [49]. Thus, although very promising for the detection of asymptomatic infections, the performance of uRDT must be further evaluated especially in areas with a already high prevalence of *pfhrp2* and *pfhrp3* gene deletions.

#### **3.2 Enzyme-linked immunosorbent assay (ELISA)**

ELISA is an enzyme-linked immunosorbent assay that is used to detect antigens and antibodies by utilizing a highly specific antibody–antigen interaction and their

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control DOI: http://dx.doi.org/10.5772/intechopen.96530*

bonding affinity to proteins on a solid surface [50]. Samples, including and positive and negative controls are inserted on a 96 well plate which enables the possibility to analyze multiple samples simultaneously, then samples get incubated and detection of the signal is generated directly or indirectly via secondary tag on the specific antibody [51]. ELISA can be very efficient diagnostic tool for *Plasmodium* because it is highly specific and sensitive and therefore can be used to screen blood donors and pregnant women who may or may not have been exposed to the parasite [1]. However there are some limitations associated with using ELISA, as it is unable to detect antibodies in patients with acute infections, patients with different *Plasmodium* species, and various antigens within the different life stages of the parasite [52]. A recent study showed a lower detection threshold (3 parasites/μl) by ELISA test of the *Plasmodium* lactate dehydrogenase (*pLDH*) as compared to RDT (50–60 parasites/μl) [53]. However, ELISA has a lower sensitivity (69.9%) to antigens than RDT (88%) [53], and can be expensive and require a trained technician to operate and interpret the results [53].

#### **3.3 Lateral flow immunoassay (LFIA)**

species and quantify parasite density, but only has a detection threshold of 10 to 50 parasites μl of blood (approximately 0.001% parasitemia, assuming an erythrocyte count of 5 106 cells μl) [33]. However, it requires well trained microscopists and is labor-intensive and time-consuming [32]. Previous studies have reported higher prevalence of asymptomatic parasitemia (6–7 times higher) using PCR compared to microscopy [5]. Nested PCR of the *18 s* rRNA genes has been commonly used to detect submicroscopic infections. PCR-based methods are also more sensitive than microscopy at detecting gametocytes particularly in asymptomatic individuals with low-density infections [34]. However, recent studies indicated a relatively high number of misdiagnosed infections [9], possibly due to low parasite density being less detectable by conventional PCR. The *18 s* rRNA gene has a reported detection thresholds of 0.5–5 parasites μl [35], but parasite density of asymptomatic infections especially in low transmission settings could be well below this threshold. As a result, many of these infections remain undetected in the general populations of several malaria-endemic countries [10, 36, 37] and provide perfect reservoirs for transmission at any time. Therefore, it is crucial to identify new gene targets or novel tools that are convenient and affordable for detecting asymptomatic infections, particularly in countries approaching elimination phase. Below are compari-

Rapid Diagnostic Test (RDT) is a lateral flow immunochromatographic test that can detect the presence of *Plasmodium* parasites by using blood from patients. The blood samples collected from patients must be lysed before the *Plasmodium* antigens being stained. RDT will indicate if the patient has malaria or not [38]. This diagnostic method is useful in rural areas as it is inexpensive and does not require expertise to perform the test [38]. There are various types of RDTs that can detect different antigens of *Plasmodium* parasites including *P. falciparum* and *P. vivax*, namely *P. falciparum* histidine-rich protein 2 (*PfHRP2*) and lactate dehydrogenase (*PfLDH*). However, recent studies indicated that parasites lacking the *pfhrp2*/*pfhrp3* genes could result in false-negative *PfHRP2*-RDT results, and these *pfhrp2*/*pfhrp3* variants have been spreading in many East and West African countries [39–42]. Reports of deletion of *HRP2* and *HRP3* genes was first reported in 2010 within the Peruvian Amazon [43] and the number of false negative RDTs has substantially increased in late 2014 [44]. Prevalence of false negative cases related to *HRP2* deletions in South America (with the exception of Peru) is lower than Asia and Sub-Saharan Africa [45].

The highest prevalence (50% *pfhrp2* deletions among all positive cases) were reported in Cambodia, Peru, and Eritrea [46]. Such a high prevalence could be explained by technical errors, mutations, selection and spread of the *pfhrp2* and 3 variants [47] from South America to Africa and Asia [41]. Recently, a novel HRP2 based Alere™ Malaria Ag P.f RDT (uRDT) was developed specifically for the detection of asymptomatic infections. Compared to the existing RDTs, the uRDT showed a greater than 10-fold improvement in diagnosing clinical and asymptomatic cases [48]. However, the sensitivities of the uRDT were inconsistent among different transmission settings [49]. Thus, although very promising for the detection of asymptomatic infections, the performance of uRDT must be further evaluated especially in areas with a already high prevalence of *pfhrp2* and *pfhrp3* gene deletions.

ELISA is an enzyme-linked immunosorbent assay that is used to detect antigens and antibodies by utilizing a highly specific antibody–antigen interaction and their

**3.2 Enzyme-linked immunosorbent assay (ELISA)**

**198**

sons of various conventional and novel detection methods.

*Current Topics and Emerging Issues in Malaria Elimination*

**3.1 Rapid diagnostic test (RDT)**

One of the current diagnostic tests in development is an antibody-based lateral flow immunoassay (LFIA). Unlike ELISA that requires repeated incubation and washing steps, LFIA is considered to be a simple, user-friendly and cost-effective method for front-line diagnosis [54]. LFIA is versatile enough to detect target genes in sample matrices including whole blood, saliva, and urine. This method is primarily applied to detecting gametocytes in malaria-infected samples. Gametocytes are the sexual form of the parasite that gets transmitted to the mosquito host. They play a very important role in malaria transmission, and can contribute to up to 80% [55] of the infectious reservoirs. Previous studies have shown that infections with parasitemia as low as 4 gametocytes/μl can sustain transmission [56]. LFIA targets *Pfs25* in infected blood samples, which is a glycophosphatidylinositol-linked protein expressed on the surface of *P. falciparum* zygotes and ookinetes but only found on female gametocytes in the human hosts [57]. The detection limit of *Pfs25* LFIA is 0.02 gametocytes/μl, much more sensitive than *pfhrp2* RDT with a detection limit of 50–100 parasites per μL of blood [35, 58]. Another protein at the surface of the female gametocyte *PSSP17*, which is presumably more abundant in saliva samples, was also investigated in Cameroon, Zambia and Sierra Leone with an estimated sensitivity of 83% (95% CI, 61 to 95) in symptomatic patients when compared to PCR as the gold standard [58]. Detection of *pfhrp2* in saliva have been reported recently using LFIA, but the sensitivity was only shown to be improved in severe to moderate form of infections with parasitemia >60,000 parasites/μL [59]. Although convenient, *pfhrp2*-based LFIA could be less useful in detecting asymptomatic infections.

#### **3.4 PCR-based methods**

The combination of PCR-based assays and sequencing technologies have revolutionized malaria diagnosis since their introduction in the early 2000s. Various gene targets have be used to monitor genetic and/or mutational changes in the parasites that cause the disease. PCR is a very common technique used in malaria diagnoses in laboratory settings. The main advantage of this technique is that it enables us to identify individuals with low parasitemia (**Table 1**). There are multiple versions of this technique including nested conventional PCR, real-time quantitative PCR and reverse transcriptase PCR. Nested PCR is the easiest and least expensive methods among the others. It only requires a thermocycler, set of primers, reagents and visualization after gel electrophoresis. It is mostly a

#### *Current Topics and Emerging Issues in Malaria Elimination*

qualitative method as it is fairly difficult to estimate parasite density on a agarose gel. Unlike conventional PCR that uses gel electrophoresis to visualize PCR products, real-time PCR or qPCR detects and quantifies the amount of amplified DNA usually by SYBR green or a fluorogenic probe designed based on a target gene segment (TaqMan). The standard cycle threshold (Ct) value is inversely proportional to the amount of target DNA in the sample [66] and allows estimation of parasite density even in submicroscopic samples. Different from nested and qPCRs, reverse transcriptase PCR uses RNA as template and transcribes RNA into complementary DNA. This method has been widely used to detect and quantify gametocyte density in malaria samples. Though PCR-based methods are undoubtedly more sensitive than microscopic diagnosis, the level of sensitivity is highly dependent on the gene targets (**Table 1**).

More importantly, there are around 20 to 150 copies of the *COX3* gene in the *Plasmodium* genome [62] and PCR analysis of this gene indicated a detection limit

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control*

demonstrated better performance than the conventional *18 s* rRNA marker in detecting low density parasite infections. There are about 250 copies of the *TARE-2* gene in the *Plasmodium* genome and this gene is highly specific to *P. falciparum* [69]. *TARE-2* has been previously shown to be useful for detecting ultra-low density *P. falciparum* infections in Papua New Guinea and Tanzania [62]. A recent study in Ghana showed a slightly higher sensitivity of *TARE-2* compared to *18 s* rRNA (81.2% vs. 80.9%) using microscopy as a gold standard, but with a lower specificity reduced by almost two-fold [37], making this gene less desirable for active case surveillance.

The recently discovered Telomere Associated Repetitive Element 2 (*TARE-2*) has

The *var* gene family is located primarily in the subtelomeric region of the *Plasmodium* genome. It is a family of genes known to be highly polymorphic. For instance, the genome of the 3D7 culture strain harbors 59 different *var* genes with an estimated 50– 150 copies per genome [14]. One of the main gene in the *var* gene family encodes the *P. falciparum* erythrocyte membrane protein 1 (*PfEMP1*) that contains a highly variable extracellular part and a well conserved intracellular *var* gene acidic terminal sequence (*var*ATS) [61]. The *var*ATS has a detection limit of 12–30 parasites in 200 μL whole blood. Though more sensitive *18 s* rRNA, *var*ATS employs a qPCR approach that could be less feasible for routine case detection in rural areas or developing countries [62].

Raman spectroscopy is a relatively novel technique that can be used to diagnose the presence of *Plasmodium*. This technique measures the amount of wavelength and light intensity of scattered light reflected from the target molecule of *Plasmodium* present in an infected sample either in liquid, gas, or solid form [70]. Raman spectroscopy can be used specifically to characterize hemozoin, a pigment produced by infected erythrocytes and can be used to track the progression of the disease and efficacy of drug treatment [71]. A specific type of Raman spectroscopy known as Surface Enhanced Raman Spectroscopy (SERS) has been utilized to detect early signs of erythrocytic infections and was shown with a detection limit of 30 parasites/μL [65]. Tip-enhanced Raman spectroscopy (TERS), on the other hand, can enhance *Plasmodium*'s hemozoin vacuole from 10<sup>6</sup> to 10<sup>7</sup> [71]. While Raman spectroscopy is more cost effectiveness than PCR and microscopy [71], this technique cannot detect hemozoin in early ring stage nor differentiate hemozoin from active and previous infections [71]. Further investigations are needed to refine Raman

signals in order to enhance the technique's detection abilities.

**4. Revolutionized techniques and genomics tools for monitoring**

Extensive level of genetic diversity observed in *Plasmodium* is a major threat to the eradication of the disease. Previous studies used sanger sequencing of the merozoite surface protein (MSP) or microsatellite genotyping methods to assess parasite genetic

of 0.6–2 parasites/μL, much more sensitive than the *18 s*-rRNA [60].

*3.4.3 Telomere associated repetitive element 2 (TARE-2)*

*DOI: http://dx.doi.org/10.5772/intechopen.96530*

*3.4.4 Var. gene acidic terminal sequence (varATS)*

**3.5 Raman spectroscopy**

**parasite changes**

**201**

#### *3.4.1 18 s rRNA*

The *18 s* rRNA genes have been the main gene target for molecular screening as it contains 5–8 copies per genome [62] in *Plasmodium*, but recent studies indicated a relatively high level of misdiagnosed infections [45]. The advantage of using the *18 s* rRNA is that it is highly specific compared to microscopy or RDT diagnoses. The primer sequences and protocols for both nested and qPCRs have been readily established. However, it fails to reveal infections with low parasite density (0.5–5 parasites/μL of blood) [35]. Thus, new target genes have been examined in the past few years with the goal to achieve an ultra-sensitive biomarker with higher sensitivity and specificity compared to the *18 s* rRNA.

#### *3.4.2 Mitochondrial cytochrome c oxidase III (COX3)*

The cytochrome c oxidase III (*COX3*) gene plays a very important role in cellular respiration [67]. It is a mitochondrial gene that inherited solely from the female gametocyte and less likely to undergo genetic recombination, making it an ideal candidate for identifying the origin and transmission of the parasites [68].


#### **Table 1.**

*Existing malaria diagnostic tests and their respective performance.*

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control DOI: http://dx.doi.org/10.5772/intechopen.96530*

More importantly, there are around 20 to 150 copies of the *COX3* gene in the *Plasmodium* genome [62] and PCR analysis of this gene indicated a detection limit of 0.6–2 parasites/μL, much more sensitive than the *18 s*-rRNA [60].

#### *3.4.3 Telomere associated repetitive element 2 (TARE-2)*

The recently discovered Telomere Associated Repetitive Element 2 (*TARE-2*) has demonstrated better performance than the conventional *18 s* rRNA marker in detecting low density parasite infections. There are about 250 copies of the *TARE-2* gene in the *Plasmodium* genome and this gene is highly specific to *P. falciparum* [69]. *TARE-2* has been previously shown to be useful for detecting ultra-low density *P. falciparum* infections in Papua New Guinea and Tanzania [62]. A recent study in Ghana showed a slightly higher sensitivity of *TARE-2* compared to *18 s* rRNA (81.2% vs. 80.9%) using microscopy as a gold standard, but with a lower specificity reduced by almost two-fold [37], making this gene less desirable for active case surveillance.

#### *3.4.4 Var. gene acidic terminal sequence (varATS)*

The *var* gene family is located primarily in the subtelomeric region of the *Plasmodium* genome. It is a family of genes known to be highly polymorphic. For instance, the genome of the 3D7 culture strain harbors 59 different *var* genes with an estimated 50– 150 copies per genome [14]. One of the main gene in the *var* gene family encodes the *P. falciparum* erythrocyte membrane protein 1 (*PfEMP1*) that contains a highly variable extracellular part and a well conserved intracellular *var* gene acidic terminal sequence (*var*ATS) [61]. The *var*ATS has a detection limit of 12–30 parasites in 200 μL whole blood. Though more sensitive *18 s* rRNA, *var*ATS employs a qPCR approach that could be less feasible for routine case detection in rural areas or developing countries [62].

#### **3.5 Raman spectroscopy**

qualitative method as it is fairly difficult to estimate parasite density on a agarose gel. Unlike conventional PCR that uses gel electrophoresis to visualize PCR products, real-time PCR or qPCR detects and quantifies the amount of amplified DNA usually by SYBR green or a fluorogenic probe designed based on a target gene segment (TaqMan). The standard cycle threshold (Ct) value is inversely proportional to the amount of target DNA in the sample [66] and allows estimation of parasite density even in submicroscopic samples. Different from nested and qPCRs, reverse transcriptase PCR uses RNA as template and transcribes RNA into complementary DNA. This method has been widely used to detect and quantify gametocyte density in malaria samples. Though PCR-based methods are undoubtedly more sensitive than microscopic diagnosis, the level of sensitivity is highly dependent on

The *18 s* rRNA genes have been the main gene target for molecular screening as it contains 5–8 copies per genome [62] in *Plasmodium*, but recent studies indicated a relatively high level of misdiagnosed infections [45]. The advantage of using the *18 s* rRNA is that it is highly specific compared to microscopy or RDT diagnoses. The primer sequences and protocols for both nested and qPCRs have been readily established. However, it fails to reveal infections with low parasite density (0.5–5 parasites/μL of blood) [35]. Thus, new target genes have been examined in the past few years with the goal to achieve an ultra-sensitive biomarker with higher sensi-

The cytochrome c oxidase III (*COX3*) gene plays a very important role in cellular respiration [67]. It is a mitochondrial gene that inherited solely from the female gametocyte and less likely to undergo genetic recombination, making it an ideal candidate for identifying the origin and transmission of the parasites [68].

RDT *HRP2*/*3* 57 99 Field 50–100 parasites/μL of blood [35] RDT *LDH* 58 93 Field 50–100 parasites/μL of blood [35] PCR *18 s* rRNA 64 92 Laboratory 0.5–5 parasites/μL of blood [60] PCR *COX3* — — Laboratory 0.6–2 parasites/μL blood [60]

PCR *TARE-2* 81 49 Laboratory 6–24 parasites in 200 μL

PCR *var*ATS — — Laboratory 12–30 parasites in 200 μL

PCR *Pfs25* — — Laboratory 0.3 mature females/μL blood [63] ELISA *LDH* 69.9 100 Laboratory 3 parasites/μL blood [53, 64] LFIA *Pfs25* — — Laboratory 0.02 gametocytes/μl blood [58] SERS Hemozoin — — Laboratory 30 parasites/μL of blood [65]

**Application Detection Limit Refs**

whole blood

whole blood

[61]

[62]

the gene targets (**Table 1**).

tivity and specificity compared to the *18 s* rRNA.

*Current Topics and Emerging Issues in Malaria Elimination*

*3.4.2 Mitochondrial cytochrome c oxidase III (COX3)*

**(%)**

*— Denotes that the sensitivity or specificity is not reported.*

*Existing malaria diagnostic tests and their respective performance.*

**Table 1.**

**200**

**Specificity (%)**

**Type Target Sensitivity**

*3.4.1 18 s rRNA*

Raman spectroscopy is a relatively novel technique that can be used to diagnose the presence of *Plasmodium*. This technique measures the amount of wavelength and light intensity of scattered light reflected from the target molecule of *Plasmodium* present in an infected sample either in liquid, gas, or solid form [70]. Raman spectroscopy can be used specifically to characterize hemozoin, a pigment produced by infected erythrocytes and can be used to track the progression of the disease and efficacy of drug treatment [71]. A specific type of Raman spectroscopy known as Surface Enhanced Raman Spectroscopy (SERS) has been utilized to detect early signs of erythrocytic infections and was shown with a detection limit of 30 parasites/μL [65]. Tip-enhanced Raman spectroscopy (TERS), on the other hand, can enhance *Plasmodium*'s hemozoin vacuole from 10<sup>6</sup> to 10<sup>7</sup> [71]. While Raman spectroscopy is more cost effectiveness than PCR and microscopy [71], this technique cannot detect hemozoin in early ring stage nor differentiate hemozoin from active and previous infections [71]. Further investigations are needed to refine Raman signals in order to enhance the technique's detection abilities.

#### **4. Revolutionized techniques and genomics tools for monitoring parasite changes**

Extensive level of genetic diversity observed in *Plasmodium* is a major threat to the eradication of the disease. Previous studies used sanger sequencing of the merozoite surface protein (MSP) or microsatellite genotyping methods to assess parasite genetic diversity. Though affordable, these methods are time-consuming and labor-intensive for large scale genetic studies. Moreover, differentiating or phasing clonal genotypes for samples with more than two clones can be difficult. Restriction enzyme cutting followed by gel electorphoresis of the MSP gene has been conventionally used to assess parasite diversity and define multiplicity of infections (MOI) based on the number of distinct bands present on a gel. MSP is the most abundant protein at the surface of the merozoite and play a critical role in the *Plasmodium* invasion mechanism to the erythrocyte [72]. It is an informative gene for resolving clonal relationships and depicting population structure of *Plasmodium* given its size polymorphisms [73]. However, in high transmission areas where parasites are highly variable and different by single nucleotide polymorphisms (SNPs), gel electorphoresis of the MSP gene would underestimate the levels of diversity and polyclonality [74]. Microsatellite genotyping has also extensively been used for *Plasmodium* population structure and genetic diversity study [75]. While this marker has the advantages of being polymorphic, evolutionary neutrally, and are abundant in the *Plasmodium* genome [76], polyclonal samples with more than two alleles detected from two or more genetic loci are usually discarded in the analyses. Thus, next-generation sequencing (NGS) technologies offer a novel, alternative approach to shed light on the polyclonal and complex nature of *Plasmodium* infections [77].

resistance, such as *crt*, *mdr1*, *dhps*, *dhfr*, and *K13* (**Table 2**) [84]. For example, a recent study based on MIPs analysis found that the *PfDHPS* gene associated with Sulfadoxine resistance has been rapidly spread from east to west of the Democratic Republic of Congo [84]. Apart from drug-resistant mutations, a number of microsatellite loci have also been added to the MIP panel to estimate genetic structure and diversity of *P. falciparum*. This technique allows multiple loci to be genotyped simultaneously. However, the design of the MIP panel would require prior infor-

K540E

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control*

Gene Crt Mdr1 Dhps Dhfr K13

**Chloroquine Amodiaquine Sulfadoxine Pyrimethamine Artemisinin**

N51I, C59R, S108N

M476I, Y493H, R539T, I543T, C580Y

Genetic information of *P. falciparum* has allowed a careful selection of gene targets for vaccine development [85]. Vaccines can offer protection against clinical malaria especially in young children and reduce transmission in a population. However, there are several challenges in developing a highly effective malaria vaccine, mostly due to the complexity in the parasite life cycle and host immune system. *Plasmodium* parasites reproduce asexually in human hosts. They can be found throughout the body's bloodstream and liver in various stages. There are numerous potential parasite antigens that elicit different levels of host immune response [86], but the protective response towards a particular antigen or one parasite life-stage is not effective in conferring protection against other stages [87]. One solution for that is to choose an immunogenic antigen/epitope that can elicit a strong immune response and potentially confer the highest efficacy. Conjugating the target antigen or epitope with an adjuvant or better drug delivery system can help elicit a stronger and safer immune response [88]. Alternatively, it is also possible to activate other immune cells such as natural killer cells and neutrophils that can elicit a long-lasting immune response [89]. Natural killer cells act faster than T cells while neutrophils can activate either a humoral or cell mediated immunity [90]. Both natural killer cells and neutrophils in response to *P. falciparum* invasion have not been studied in depth compared to antibodies and T cells [90]. To date, there are three types of malarial vaccines that are being studied and tested in clinical trials: pre-erythrocytic,

mation of the gene regions of interest.

**Antimalarial**

**Table 2.**

**5. From parasite genes to malaria vaccines**

Mutation(s) K76T N86Y, Y184F A437G,

*DOI: http://dx.doi.org/10.5772/intechopen.96530*

*Gene mutations associated with antimalarial drug resistance.*

erythrocytic, and transmission blocking vaccines (**Table 3**).

Pre-erythrocytic vaccines aim to kill infected hepatocytes and prevent sporozoites from reaching the liver [101]. There are various gene targets currently being

RTS,S/AS01E is a well-known pre-erythrocytic vaccine that has successfully completed phase III of clinical testing. It is currently distributed by World Health

**5.1 Pre-erythrocytic vaccines**

investigated and in clinical trials.

*5.1.1 RTS,S/AS01E*

**203**

#### **4.1 Amplicon deep sequencing**

In regions in high malaria endemicity, individuals typically harbor multiple *Plasmodium falciparum* isolates due to repeated exposure to mosquitoes infected with multiple parasite isolates [78]. Polyclonal infections have become a growing concern as some parasite isolates may be resistant to antimalarial drugs and/or more pathogenic [79]. Identification of genetically distinct clones is necessary to critically evaluate the causation of resistance to drugs and any other therapeutic treatments. Recently, due to reduced cost of high throughput NGS technology, other types of molecular tools have emerged to address the complex issue related to MOI. MOI is defined as the number of parasite clones within an infected sample. Deep sequencing of a targeted gene amplicon coupled with bioinformatic analyses allow differentiation of various *P. falciparum* strains based on SNPs [75] and discrimination of major from minor clones [80]. It also provides an increased capability to detect the genetic relatedness among clones within and between hosts as well as minor *P. falciparum* variants [81]. This technique can be applied to different gene regions such as Circumsporozoite (CSP), MSP1/2, and Apical membrane antigen (AMA) to monitor changes as well as selection pressure acting on the parasite populations [82].

#### **4.2 Molecular inversion probe**

Molecular inversion probes (MIPs) are another deep sequencing technique that targets several short gene regions across the genome. MIPs are single stranded DNA molecules that contain flanking regions of the targeted gene regions up to several hundred base pairs long [83]. MIPs can hybridize with the target sequence and undergo gap filling ligation to form circular DNA. The target sequence will also contain adaptors and barcodes to be further amplified by PCR [83]. The advantages of this technique include low rate of errors, small amount of DNA samples, high throughput, and cost-effective for several hundreds of samples. It is scalable to the number of targeted gene regions and samples, requires minimal costs in terms of reagents and labor, and allows efficient capture of DNA extracted from dried blood spots [84]. The latter advantage would make large-scale population studies feasible. MIPs have been used for monitoring SNP mutations associated with drug

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control DOI: http://dx.doi.org/10.5772/intechopen.96530*


**Table 2.**

diversity. Though affordable, these methods are time-consuming and labor-intensive for large scale genetic studies. Moreover, differentiating or phasing clonal genotypes for samples with more than two clones can be difficult. Restriction enzyme cutting followed by gel electorphoresis of the MSP gene has been conventionally used to assess parasite diversity and define multiplicity of infections (MOI) based on the number of distinct bands present on a gel. MSP is the most abundant protein at the surface of the merozoite and play a critical role in the *Plasmodium* invasion mechanism to the erythrocyte [72]. It is an informative gene for resolving clonal relationships and depicting population structure of *Plasmodium* given its size polymorphisms [73]. However, in high transmission areas where parasites are highly variable and different by single nucleotide polymorphisms (SNPs), gel electorphoresis of the MSP gene would underestimate the levels of diversity and polyclonality [74]. Microsatellite genotyping has also extensively been used for *Plasmodium* population structure and genetic diversity study [75]. While this marker has the advantages of being polymorphic, evolutionary neutrally, and are abundant in the *Plasmodium* genome [76], polyclonal samples with more than two alleles detected from two or more genetic loci are usually discarded in the analyses. Thus, next-generation sequencing (NGS) technologies offer a novel, alternative approach to shed light on the polyclonal

In regions in high malaria endemicity, individuals typically harbor multiple *Plasmodium falciparum* isolates due to repeated exposure to mosquitoes infected with multiple parasite isolates [78]. Polyclonal infections have become a growing concern as some parasite isolates may be resistant to antimalarial drugs and/or more pathogenic [79]. Identification of genetically distinct clones is necessary to critically evalu-

ate the causation of resistance to drugs and any other therapeutic treatments. Recently, due to reduced cost of high throughput NGS technology, other types of molecular tools have emerged to address the complex issue related to MOI. MOI is defined as the number of parasite clones within an infected sample. Deep sequencing of a targeted gene amplicon coupled with bioinformatic analyses allow differentiation of various *P. falciparum* strains based on SNPs [75] and discrimination of major from minor clones [80]. It also provides an increased capability to detect the genetic relatedness among clones within and between hosts as well as minor *P. falciparum* variants [81]. This technique can be applied to different gene regions such as Circumsporozoite (CSP), MSP1/2, and Apical membrane antigen (AMA) to monitor

changes as well as selection pressure acting on the parasite populations [82].

MIPs have been used for monitoring SNP mutations associated with drug

Molecular inversion probes (MIPs) are another deep sequencing technique that targets several short gene regions across the genome. MIPs are single stranded DNA molecules that contain flanking regions of the targeted gene regions up to several hundred base pairs long [83]. MIPs can hybridize with the target sequence and undergo gap filling ligation to form circular DNA. The target sequence will also contain adaptors and barcodes to be further amplified by PCR [83]. The advantages of this technique include low rate of errors, small amount of DNA samples, high throughput, and cost-effective for several hundreds of samples. It is scalable to the number of targeted gene regions and samples, requires minimal costs in terms of reagents and labor, and allows efficient capture of DNA extracted from dried blood spots [84]. The latter advantage would make large-scale population studies feasible.

and complex nature of *Plasmodium* infections [77].

*Current Topics and Emerging Issues in Malaria Elimination*

**4.1 Amplicon deep sequencing**

**4.2 Molecular inversion probe**

**202**

*Gene mutations associated with antimalarial drug resistance.*

resistance, such as *crt*, *mdr1*, *dhps*, *dhfr*, and *K13* (**Table 2**) [84]. For example, a recent study based on MIPs analysis found that the *PfDHPS* gene associated with Sulfadoxine resistance has been rapidly spread from east to west of the Democratic Republic of Congo [84]. Apart from drug-resistant mutations, a number of microsatellite loci have also been added to the MIP panel to estimate genetic structure and diversity of *P. falciparum*. This technique allows multiple loci to be genotyped simultaneously. However, the design of the MIP panel would require prior information of the gene regions of interest.

#### **5. From parasite genes to malaria vaccines**

Genetic information of *P. falciparum* has allowed a careful selection of gene targets for vaccine development [85]. Vaccines can offer protection against clinical malaria especially in young children and reduce transmission in a population. However, there are several challenges in developing a highly effective malaria vaccine, mostly due to the complexity in the parasite life cycle and host immune system. *Plasmodium* parasites reproduce asexually in human hosts. They can be found throughout the body's bloodstream and liver in various stages. There are numerous potential parasite antigens that elicit different levels of host immune response [86], but the protective response towards a particular antigen or one parasite life-stage is not effective in conferring protection against other stages [87]. One solution for that is to choose an immunogenic antigen/epitope that can elicit a strong immune response and potentially confer the highest efficacy. Conjugating the target antigen or epitope with an adjuvant or better drug delivery system can help elicit a stronger and safer immune response [88]. Alternatively, it is also possible to activate other immune cells such as natural killer cells and neutrophils that can elicit a long-lasting immune response [89]. Natural killer cells act faster than T cells while neutrophils can activate either a humoral or cell mediated immunity [90]. Both natural killer cells and neutrophils in response to *P. falciparum* invasion have not been studied in depth compared to antibodies and T cells [90]. To date, there are three types of malarial vaccines that are being studied and tested in clinical trials: pre-erythrocytic, erythrocytic, and transmission blocking vaccines (**Table 3**).

#### **5.1 Pre-erythrocytic vaccines**

Pre-erythrocytic vaccines aim to kill infected hepatocytes and prevent sporozoites from reaching the liver [101]. There are various gene targets currently being investigated and in clinical trials.

#### *5.1.1 RTS,S/AS01E*

RTS,S/AS01E is a well-known pre-erythrocytic vaccine that has successfully completed phase III of clinical testing. It is currently distributed by World Health


*P. falciparum*. CSP are surface proteins that is important in hepatocyte invasion. Interactions between CSP and heparin sulfate proteoglycans (HSPGs) allows the sporozoite to attach to the surface of hepatocytes and triggers a signaling cascade that allows for the sporozoite to invade liver cells [102]. Subsequent to invasion, the sporozoites will mature, multiply, and feed on the hepatocytes until the hepatocytes lyse and release merozoites into the bloodstream. Preventing the invasion of sporozoites will inhibit the progression and severity of the disease. Using the C-terminus and central tandem repeat (NANP) of *PfCSP*, Hepatitis B surface membrane antigen (HSbAg), and an AS01 adjuvant system, the vaccine will elicit a strong, stable immune response [91]. After vaccination, the host immune system will response to *PfCSP* antigen by producing anti-CSP antibodies and activating CD4+ T cells [103]. RTS,S/AS01E is given on a three-dose schedule within three months followed by a fourth dose at 20 months [91]. It has been shown across clinical malarial studies that the vaccine has an 39–50% efficacy in children ages 5–17 months and 23–30% efficacy in children ages 6–12 months [104–106]. Efficacy warned rapidly from 35.2% and 20.3% to 19.1% and 12.7%, respectively, in children and infants within 20–32 months without a booster [91]. Such a decay in vaccine efficacy could be due to reduced IgG and IgM antibodies against CSP antigen. Anti-CSP antibodies were shown to increase by almost 10-fold from 318.2 EU/mL to 34.2 EU/mL in children one month after a booster was given [96]. Apart from the short-lived nature of the vaccine, older children who were vaccinated showed an increased risk of malarial infections, likely due to RTS,S/AS01E interference with naturally acquired immunity [96]. Another similar vaccine R21 is currently testing in Phase 1/2a clinical trials [107]. This vaccine aims to elicit a high anti-CSP antibody content similar to the mechanisms of RTS,S vaccine. R21 comprises particles from CSP-HBsAg protein infused with an adjuvant, matrix-M [107]. It has been shown to increase the production of T cells and is still in the process of development [96]. Further studies should investigate alternative antigens that can elicit stronger and long-lasting efficacy as well as the mechanisms of the cell-mediated immune response against

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control*

*DOI: http://dx.doi.org/10.5772/intechopen.96530*

*Plasmodium falciparum* sporozoites (PfSPZ) is another pre-erythrocytic vaccine target. As aforementioned, sporozoites transferred from the infected Anopheles mosquito to the human host. They enter the bloodstream and reach the liver before invading the hepatocytes using the sporozoite proteins P36 and P52. P36 interacts

parasitophorous vacuole that facilitates hepatocyte invasion [108]. *PfSPZ* vaccine prevents the sporozoites reaching the liver and infecting hepatocytes [109], and is currently undergoing phase III clinical trial [92]. *PfSPZ* vaccine was designed to have a live, whole sporozoite that is radiated-attenuated. It is injected intravenously and given in 3–5 doses. Recent studies using controlled human malaria infection (CHMI) showed that this vaccine provided about 33 weeks of stabilized protection in 50% of the vaccinated subjects [110]. The vaccine induced interferon gamma (IFN-γ) that can recruit and activate CD8+ and CD4+ T cells against homologous and heterologous parasitic strains [110]. Subjects who received the *PfSPZ* vaccine also developed IgM antibodies that can help inhibit proliferation of the parasites [111]. However, the vaccine requires specific storage in ultra-cold condition and trained medical workers to inject the vaccine intravenously [96]. Furthermore, efficacy varied by locations. For example, in Mali, 29% efficacy was reported in subject who were exposed to heterologous strain; whereas in CHMI, 83% of subjects were found to be protected from exposure to heterologous strains [92]. Further

with hepatocyte's extracellular receptor EphA2 to create a protective

malaria in humans.

*5.1.2 PfSPZ*

**205**

#### **Table 3.**

*Overview of current vaccines candidates against* Plasmodium falciparum*.*

Organization (WHO) in Malawi, Ghana, and Kenya to further investigate vaccine efficacy [96]. RTS,S is a vaccine that targets circumsporozoite proteins (CSP) from *Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control DOI: http://dx.doi.org/10.5772/intechopen.96530*

*P. falciparum*. CSP are surface proteins that is important in hepatocyte invasion. Interactions between CSP and heparin sulfate proteoglycans (HSPGs) allows the sporozoite to attach to the surface of hepatocytes and triggers a signaling cascade that allows for the sporozoite to invade liver cells [102]. Subsequent to invasion, the sporozoites will mature, multiply, and feed on the hepatocytes until the hepatocytes lyse and release merozoites into the bloodstream. Preventing the invasion of sporozoites will inhibit the progression and severity of the disease. Using the C-terminus and central tandem repeat (NANP) of *PfCSP*, Hepatitis B surface membrane antigen (HSbAg), and an AS01 adjuvant system, the vaccine will elicit a strong, stable immune response [91]. After vaccination, the host immune system will response to *PfCSP* antigen by producing anti-CSP antibodies and activating CD4+ T cells [103]. RTS,S/AS01E is given on a three-dose schedule within three months followed by a fourth dose at 20 months [91]. It has been shown across clinical malarial studies that the vaccine has an 39–50% efficacy in children ages 5–17 months and 23–30% efficacy in children ages 6–12 months [104–106]. Efficacy warned rapidly from 35.2% and 20.3% to 19.1% and 12.7%, respectively, in children and infants within 20–32 months without a booster [91]. Such a decay in vaccine efficacy could be due to reduced IgG and IgM antibodies against CSP antigen. Anti-CSP antibodies were shown to increase by almost 10-fold from 318.2 EU/mL to 34.2 EU/mL in children one month after a booster was given [96]. Apart from the short-lived nature of the vaccine, older children who were vaccinated showed an increased risk of malarial infections, likely due to RTS,S/AS01E interference with naturally acquired immunity [96]. Another similar vaccine R21 is currently testing in Phase 1/2a clinical trials [107]. This vaccine aims to elicit a high anti-CSP antibody content similar to the mechanisms of RTS,S vaccine. R21 comprises particles from CSP-HBsAg protein infused with an adjuvant, matrix-M [107]. It has been shown to increase the production of T cells and is still in the process of development [96]. Further studies should investigate alternative antigens that can elicit stronger and long-lasting efficacy as well as the mechanisms of the cell-mediated immune response against malaria in humans.

#### *5.1.2 PfSPZ*

*Plasmodium falciparum* sporozoites (PfSPZ) is another pre-erythrocytic vaccine target. As aforementioned, sporozoites transferred from the infected Anopheles mosquito to the human host. They enter the bloodstream and reach the liver before invading the hepatocytes using the sporozoite proteins P36 and P52. P36 interacts with hepatocyte's extracellular receptor EphA2 to create a protective parasitophorous vacuole that facilitates hepatocyte invasion [108]. *PfSPZ* vaccine prevents the sporozoites reaching the liver and infecting hepatocytes [109], and is currently undergoing phase III clinical trial [92]. *PfSPZ* vaccine was designed to have a live, whole sporozoite that is radiated-attenuated. It is injected intravenously and given in 3–5 doses. Recent studies using controlled human malaria infection (CHMI) showed that this vaccine provided about 33 weeks of stabilized protection in 50% of the vaccinated subjects [110]. The vaccine induced interferon gamma (IFN-γ) that can recruit and activate CD8+ and CD4+ T cells against homologous and heterologous parasitic strains [110]. Subjects who received the *PfSPZ* vaccine also developed IgM antibodies that can help inhibit proliferation of the parasites [111]. However, the vaccine requires specific storage in ultra-cold condition and trained medical workers to inject the vaccine intravenously [96]. Furthermore, efficacy varied by locations. For example, in Mali, 29% efficacy was reported in subject who were exposed to heterologous strain; whereas in CHMI, 83% of subjects were found to be protected from exposure to heterologous strains [92]. Further

Organization (WHO) in Malawi, Ghana, and Kenya to further investigate vaccine efficacy [96]. RTS,S is a vaccine that targets circumsporozoite proteins (CSP) from

**Name of Vaccine**

RTS,S/ AS01E

*PfLSA* Vaccine

*MSP1* Vaccine

*AMA-1* Vaccine

R21 Vaccine

*Pfs 230* & *Pfs48/45*

*PfSPZ* Live,

**Type of Vaccine**

Recombinant protein vaccine

radiationattenuated vaccine

Recombinant protein vaccine

Recombinant protein vaccine?

Recombinant protein vaccines

Potential new target

*Pfs25* Recombinant conjugated vaccines

*PfGARP* Potential new target

> Potential new target

*PfRH5*- *PfCyRPA*-*PfRipr* (RCR) complex

**Table 3.**

**204**

Subunit based Vaccine **Target of Vaccine Malarial Life**

Circumsporozoite protein (CSP)

*Current Topics and Emerging Issues in Malaria Elimination*

Sporozoite (SPZ) Pre-

Liver-stage antigen 1

Illicit a higher anti-CSP antibody count

*Pfs230* and *Pfs48/45* combined with a FAB fragment of a monoclonal antibody, 4F12, can act on parasitic gametes by forming a membrane-bound protein complex and increase the vaccine

activity

host.

protein

Antibodies against the *Pfs25* antigen in the human host to stop the development and transmission of the parasite when the vector feeds on the

Glutamic acid rich

Induce straintranscending neutralizing antibodies against blood-stage *P. falciparum*

*Overview of current vaccines candidates against* Plasmodium falciparum*.*

(*LSA-1*)

**Cycle Stage**

Erythrocytic

MSP1 Erythrocytic Phase I Efficacy has yet

*AMA-1* Erythrocytic Phase II Efficacy has yet

Erythrocytic Phase

Transmission Blocking Vaccine

Transmission Blocking Vaccine

Erythrocytic Phase

I/IIa

Pre-Erythrocytic

Pre-Erythrocytic **Phase of Vaccine**

Phase III

Phase III

**Efficacy Refs**

[91]

[92]

[93]

[94]

[95]

[96]

[97]

[98]

[99]

[100]

35.2% in Children 20.3% in Infants

Homologous challenge: 64% Heterologous challenge: 83%

to be found in humans

to be found in humans

Efficacy has yet to be found in humans

None Efficacy has yet to be found in humans

Phase I Efficacy has yet to be found in humans

> to be found in humans

Efficacy has yet to be found in humans

Erythrocytic None Efficacy has yet

I/II

Phase I Efficacy has yet to be found in humans

studies are needed to compare efficacy among different geographical or transmission settings.

*5.2.2 PfAMA-1*

*DOI: http://dx.doi.org/10.5772/intechopen.96530*

*5.2.3 PfGARP*

**207**

up with the AMA1 protein to increase efficacy.

was reduced by 76–87% *in vitro* [99].

*5.2.4 PfRH5-PfCyRPA-PfRipr (RCR) complex*

The erythrocytic vaccine based on Apical Membrane Antigen 1 (AMA1) is currently in phase II clinical trial [116]. The AMA1 protein is approximately 83 kDa and can be found in both the merozoite and sporozoite stages of *P. falciparum* [116]. The complex AMA1 and another parasite protein namely the rhoptry neck protein 2 (RON2), is essential for merozoite invasion during the blood stage of infection, and initiates the parasite traversal into the RBCs [117]. The AMA1 vaccine has been shown to elicit high levels of antibodies that can block the invasion of the erythrocytes, despite high polymorphisms observed in the AMA1 protein [117]. The AMA1- Diversity Covering (DiCo) vaccine was thus designed to include three recombinant variants of AMA1 and this vaccine is currently testing in phase Ia/Ib clinical trial [118]. To increase efficacy of the AMA1 vaccine, AMA1 was paired with the RON2 receptor and vaccinated in eight *Aotus* monkeys [117]. Half of the monkeys were able to achieve complete immunity from the *P. falciparum* infection when vaccinated with AMA1 and RON2 [117]. The vaccine with AMA1 and RON2 induced a higher level of antibodies than the AMA1 vaccine. Further investigation is needed to examine other potential AMA1 variants or merozoite structures that can be paired

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control*

*Plasmodium falciparum* glutamic acid rich protein (PfGARP) is an 80 kDa antigen commonly expressed on the surface of infected erythrocytes during the late trophozoite stage [119]. The *PfGARP* gene is relatively conserved. Antibodies against the *PfGARP* antigen protein have been shown to confer protection against severe malaria and reduce parasite densities by 3.5 folds [99]. Further, anti-*PfGARP* antibodies were able to successfully induce apoptosis in ring-stage parasite cultures, resulting in full loss of their mitochondrial function within a 24 hour period [99]. In addition, the size of food vacuoles in the parasites was decreased or condensed tightly around the hemozoin crystals, making them inaccessible and parasite growth

The *PfRH5*-*PfCyRPA*-*PfRipr* (RCR) complex is a protein trimer composed of three different proteins *PfRH5*, *PfCyRPA*, and *PfRipr* that are found on the surface of merozoites [100]. *P. falciparum* Reticulocyte-binding Protein Homolog 5 (*PfRH5*) is a 63 kDa protein commonly expressed during the schizont stage. After *PfRH5* binds with basigin (a receptor found on human erythrocytes), a large amount of calcium is released to initiate invasion [100]. Monoclonal antibodies against *PfRH5* has been found to disrupt the binding between basigin and *PfRH5* [100]. *P. falciparum* Cysteine-rich Protective Antigen (*PfCyRPA*) is a highly conserved 43 kDa protein [100]. It plays an important role in erythrocyte invasion by interacting with *PfRH5* to bind to the receptor basigin [56]. Although *PfCyRPA* is not immunogenic compared to *PfRipr* and *PfRH5*, monoclonal antibodies produced against this protein can cause cross strain neutralization [100]. *P. falciparum* RH5 interacting Protein (*PfRipr*) is a highly conserved 120 kDa protein found in the schizont stage [100]. It is composed of 87 cysteines and 10 epidermal growth factorlike (EGF) domains [100]. Prior to erythrocyte invasion, *PfRipr* cleaves into two different fragments including the N-terminus and C-terminus. The N-terminus contains EGF domains 1 and 2 while the C-terminus contains EGF domains 3–10 [100]. Antibodies against *PfRipr* EGF domains 6–8 have been shown to neutralize

#### *5.1.3 PfLSA-1 and PfLSA-3*

Liver surface antigen (LSA) is another pre-erythrocytic vaccine target. LSA is essential for the survival of the parasites during the late liver schizogony stage [93]. Schizonts are asexual stage of *Plasmodium* that developed from sporozoites and matured in infected hepatocytes. Once the infected hepatocytes are filled up with mature schizonts, they rupture and release merozoites into the bloodstream to invade erythrocytes. LSA vaccine prevents the maturation of schizonts and rupture of infected hepatocytes. There are two liver stage antigens, LSA-1 and LSA-3, that are used as vaccine targets. *PfLSA-1* is high conserved in *P. falciparum* and is found in parasitophorous vacuole in the liver stage of the parasites. *PfLSA-1* contains 17 amino acids repeats and is associated with the late liver schizont stage [93, 112]. LSA-1 can induce IgG and IgM antibodies as well as CD4+ T cell production [93, 113]. LSA-1 vaccine is currently in phase I clinical trial and is still unclear its efficacy in humans. LSA-3, on the other hand, is found in dense granules in the blood stage of the parasites and the protein is about 175 kDa [114]. LSA-3 appears to play a role in the parasitic growth in infected hepatocytes and erythrocytes [114]. A recent study indicated that LSA-3 provided full protect to chimpanzees from heterologous *P. falciparum* sporozoites [93]. Like LSA-1, LSA-3 is also in phase I clinical trial and its efficacy and immunogenicity in humans remain unclear.

#### **5.2 Erythrocytic vaccines**

Erythrocytic vaccines aim to kill and terminate the asexual reproduction and invasion of the parasite within red blood cells (RBC). Infected individuals typically experience symptoms when the parasites invade a threshold number of RBCs and disrupt their normal functions. Preventing the parasites developed into blood stage will inhibit progression of malaria symptoms such as chills, aches, and fevers.

#### *5.2.1 PfMSP1*

Merozoite surface protein 1 (MSP1) is one of the targets used for erythrocytic vaccine. MSP1 is a glycosylphosphatidylinositol-anchored protein found in abundance on the surface of the merozoites [115]. MSP1 plays an important role in the invasion of erythrocytes as it binds and recruits other peripheral merozoite surface proteins to form a complex [115]. MSP1 starts off as a precursor of about 196 kDa and then cleave into four subunits before invading the erythrocytes [94]. The four subunits are held non-covalently forming a complex attached to the merozoite's GPI anchor [94]. Once the MSP1 complex is formed, it binds with the receptors on erythrocytes and activates a spectrin-binding function to enter the erythrocytes [94]. MSP1 has been shown to elicit both humoral (IgM and IgG antibodies) and cell-mediated immune responses (memory T cells) that lasted about 6 months after immunization [94]. These results are promising as MSP1 vaccine will also activate antibodies for complement fixation, induce opsonizing antibodies, and initiate secretion of reactive oxygen species by other immune cells [94]. To date, MSP1 vaccine is in phase I clinical trial. Further investigation is needed to evaluate efficacy.

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control DOI: http://dx.doi.org/10.5772/intechopen.96530*

#### *5.2.2 PfAMA-1*

studies are needed to compare efficacy among different geographical or transmis-

Liver surface antigen (LSA) is another pre-erythrocytic vaccine target. LSA is essential for the survival of the parasites during the late liver schizogony stage [93]. Schizonts are asexual stage of *Plasmodium* that developed from sporozoites and matured in infected hepatocytes. Once the infected hepatocytes are filled up with mature schizonts, they rupture and release merozoites into the bloodstream to invade erythrocytes. LSA vaccine prevents the maturation of schizonts and rupture of infected hepatocytes. There are two liver stage antigens, LSA-1 and LSA-3, that are used as vaccine targets. *PfLSA-1* is high conserved in *P. falciparum* and is found in parasitophorous vacuole in the liver stage of the parasites. *PfLSA-1* contains 17 amino acids repeats and is associated with the late liver schizont stage [93, 112]. LSA-1 can induce IgG and IgM antibodies as well as CD4+ T cell production [93, 113]. LSA-1 vaccine is currently in phase I clinical trial and is still unclear its efficacy in humans. LSA-3, on the other hand, is found in dense granules in the blood stage of the parasites and the protein is about 175 kDa [114]. LSA-3 appears to play a role in the parasitic growth in infected hepatocytes and erythrocytes [114]. A recent study indicated that LSA-3 provided full protect to chimpanzees from heterologous *P. falciparum* sporozoites [93]. Like LSA-1, LSA-3 is also in phase I clinical trial and its efficacy and immunogenicity in humans remain

Erythrocytic vaccines aim to kill and terminate the asexual reproduction and invasion of the parasite within red blood cells (RBC). Infected individuals typically experience symptoms when the parasites invade a threshold number of RBCs and disrupt their normal functions. Preventing the parasites developed into blood stage will inhibit progression of malaria symptoms such as chills, aches, and

Merozoite surface protein 1 (MSP1) is one of the targets used for erythrocytic vaccine. MSP1 is a glycosylphosphatidylinositol-anchored protein found in abundance on the surface of the merozoites [115]. MSP1 plays an important role in the invasion of erythrocytes as it binds and recruits other peripheral merozoite surface proteins to form a complex [115]. MSP1 starts off as a precursor of about 196 kDa and then cleave into four subunits before invading the erythrocytes [94]. The four subunits are held non-covalently forming a complex attached to the merozoite's GPI anchor [94]. Once the MSP1 complex is formed, it binds with the receptors on erythrocytes and activates a spectrin-binding function to enter the erythrocytes [94]. MSP1 has been shown to elicit both humoral (IgM and IgG antibodies) and cell-mediated immune responses (memory T cells) that lasted about 6 months after immunization [94]. These results are promising as MSP1 vaccine will also activate antibodies for complement fixation, induce opsonizing antibodies, and initiate secretion of reactive oxygen species by other immune cells [94]. To date,

MSP1 vaccine is in phase I clinical trial. Further investigation is needed to evaluate

sion settings.

unclear.

fevers.

*5.2.1 PfMSP1*

efficacy.

**206**

**5.2 Erythrocytic vaccines**

*5.1.3 PfLSA-1 and PfLSA-3*

*Current Topics and Emerging Issues in Malaria Elimination*

The erythrocytic vaccine based on Apical Membrane Antigen 1 (AMA1) is currently in phase II clinical trial [116]. The AMA1 protein is approximately 83 kDa and can be found in both the merozoite and sporozoite stages of *P. falciparum* [116]. The complex AMA1 and another parasite protein namely the rhoptry neck protein 2 (RON2), is essential for merozoite invasion during the blood stage of infection, and initiates the parasite traversal into the RBCs [117]. The AMA1 vaccine has been shown to elicit high levels of antibodies that can block the invasion of the erythrocytes, despite high polymorphisms observed in the AMA1 protein [117]. The AMA1- Diversity Covering (DiCo) vaccine was thus designed to include three recombinant variants of AMA1 and this vaccine is currently testing in phase Ia/Ib clinical trial [118]. To increase efficacy of the AMA1 vaccine, AMA1 was paired with the RON2 receptor and vaccinated in eight *Aotus* monkeys [117]. Half of the monkeys were able to achieve complete immunity from the *P. falciparum* infection when vaccinated with AMA1 and RON2 [117]. The vaccine with AMA1 and RON2 induced a higher level of antibodies than the AMA1 vaccine. Further investigation is needed to examine other potential AMA1 variants or merozoite structures that can be paired up with the AMA1 protein to increase efficacy.

#### *5.2.3 PfGARP*

*Plasmodium falciparum* glutamic acid rich protein (PfGARP) is an 80 kDa antigen commonly expressed on the surface of infected erythrocytes during the late trophozoite stage [119]. The *PfGARP* gene is relatively conserved. Antibodies against the *PfGARP* antigen protein have been shown to confer protection against severe malaria and reduce parasite densities by 3.5 folds [99]. Further, anti-*PfGARP* antibodies were able to successfully induce apoptosis in ring-stage parasite cultures, resulting in full loss of their mitochondrial function within a 24 hour period [99]. In addition, the size of food vacuoles in the parasites was decreased or condensed tightly around the hemozoin crystals, making them inaccessible and parasite growth was reduced by 76–87% *in vitro* [99].

#### *5.2.4 PfRH5-PfCyRPA-PfRipr (RCR) complex*

The *PfRH5*-*PfCyRPA*-*PfRipr* (RCR) complex is a protein trimer composed of three different proteins *PfRH5*, *PfCyRPA*, and *PfRipr* that are found on the surface of merozoites [100]. *P. falciparum* Reticulocyte-binding Protein Homolog 5 (*PfRH5*) is a 63 kDa protein commonly expressed during the schizont stage. After *PfRH5* binds with basigin (a receptor found on human erythrocytes), a large amount of calcium is released to initiate invasion [100]. Monoclonal antibodies against *PfRH5* has been found to disrupt the binding between basigin and *PfRH5* [100]. *P. falciparum* Cysteine-rich Protective Antigen (*PfCyRPA*) is a highly conserved 43 kDa protein [100]. It plays an important role in erythrocyte invasion by interacting with *PfRH5* to bind to the receptor basigin [56]. Although *PfCyRPA* is not immunogenic compared to *PfRipr* and *PfRH5*, monoclonal antibodies produced against this protein can cause cross strain neutralization [100]. *P. falciparum* RH5 interacting Protein (*PfRipr*) is a highly conserved 120 kDa protein found in the schizont stage [100]. It is composed of 87 cysteines and 10 epidermal growth factorlike (EGF) domains [100]. Prior to erythrocyte invasion, *PfRipr* cleaves into two different fragments including the N-terminus and C-terminus. The N-terminus contains EGF domains 1 and 2 while the C-terminus contains EGF domains 3–10 [100]. Antibodies against *PfRipr* EGF domains 6–8 have been shown to neutralize

the parasites [100]. Combining the anti-PfCyRPA c12 mAb with anti-RH5 BS1.2 mAbr will inhibit parasite growth *in vitro* from 21–31% to 59%. Thus, this antigen protein complex is expected to elicit a strong immune response against blood stage *P. falciparum* [120].

as tracking and stopping transmission of the disease. Several hundreds of

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control*

parasite reservoirs and effectively control transmission.

*DOI: http://dx.doi.org/10.5772/intechopen.96530*

The authors declare no conflict of interest.

**Acknowledgements**

**Conflict of interest**

**Author contributions**

**Funding**

**209**

*P. falciparum* genomes have been generated in the past two decades. Genetic variation and function of various genes have improved our understanding of mutational changes, molecular structure, and evolutionary mechanisms in *Plasmodium*. There is an urgent need to retrieve utmost biological meaning from the available genomic data and translate such into tools that help resolve epidemiological challenges. This includes the identification of novel antigens for accurate and affordable diagnostic assays and vaccines, informative biomarkers that can distinguish different isolates and pinpoint the source of infections at fine geographical scale, and sensitive tool(s) for large-scale screening of asymptomatic infections in both high and low transmission areas. Future studies should examine how climate/environmental changes and selective pressure from interventions mediate genetic changes in the parasites, how host immune system responses to parasite changes, and how to uncover hidden

We thank our colleagues at the University of North Carolina at Charlotte and the University of Ghana for the inspiration and discussion that lead to the conceptualization of this paper. We also thank the reviewers for their insightful comments.

Conceptualization, C.C.D. and E.L.; resources, D.A.J. and E.L.; writing—original draft preparation, C.C.D., C.T.F., J.H., and E.L.; writing—review and editing, C.C. D., C.T.F., L.E.A, Y.A.A., D.A.J., and and E.L.; funding acquisition, E.L. All authors

This research was funded by NIH/NIAID, grant number R15 AI138002.

have read and agreed to the published version of the manuscript.

#### **5.3 Transmission blocking vaccine (TBV)**

TBV aims to prevent and kill the sexual stages of the *Plasmodium* parasites before transferring into the mosquito hosts. This vaccine offers protection against infection and transmission.

#### *5.3.1 Pfs25*

*Pfs25* is an important glycophosphatidylinositol-linked protein expressed on the surface of ookinetes. It is found only within the *Anopheles* host and is approximately 25 kDa with 11 disulfie bonds [121]. The parasites require *Pfs25* to survive in the *Anopheles*'s midgut and develop into oocysts [122]. *Pfs25* is a conserve protein with low diversity. *Pfs25* vaccine was designed to elicit antibodies against the *Pfs25* antigen in humans and prohibit the development and transmission of gametocytes [98]. However, a recent study based on an adjuvant of *Pfs25* and a non-enveloped virus like protein (VLP) indicated weak IgG antibody responses in healthy individuals [123]. In another study, the antibody response of *Pfs25* proteins combined with four different adjuvants including alum, Toll-like receptor 4 (TLR-4) agonist glucopyranosal lipid A (GLA) plus alum, squalene–oil-in-water emulsion, and GLA plus squalene–oil-in-water emulsion were compared in mice. *Pfs25* combined with GLA plus squalene–oil-in-water emulsion was shown to induce the highest amounts of IgG antibodies [124]. Further studies should examine the formulation of this vaccine for better efficacy.

#### *5.3.2 Pfs230 and Pfs48/45*

*Pfs230* and *Pfs48/45*-based vaccines are antigens rich in cysteine produced by the sexual stage gametocytes [97]. *Pfs48/45* can be found on the plasma membrane surface of both male and female gametocytes and are bounded to a GPI anchor that form a complex with *Pfs230* [125]. *Pfs230* is a 230 kDa protein that contains 14 6 cysteine rich domains [97]. *Pfs48/45*, on the other hand, contains three 6-cysteine rich domains [125]. The 6-cysteine rich domains of these antigens are essential for the formation of disulfide bonds on epitopes needed for antibody elicitation [125]. A recent study showed that mice injected with fragments of *Pfs48/45* and *Pfs230* prodomain produced higher levels of antibodies that induced complement fixation [125]. *Pfs230* and *Pfs48/45* combined with a FAB fragment of a monoclonal antibody 4F12 have been shown to further increase vaccine efficacy [97]. This vaccine is currently in early clinical development phase [97, 125].

#### **6. Conclusion**

WHO aims to achieve malaria elimination in at least 35 countries, reduce incidence and mortality rates by 90%, and prevent resurgence in malaria-free countries by 2030. This ambitious goal has been challenged by the emergence and spread of antimalarial resistance, inaccurate diagnostic testing, asymptomatic transmission, and lack of effective vaccines [126]. Information of the *Plasmodium* genomes allow us to improve and reinvent tools/techniques for monitoring parasite changes as well *Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control DOI: http://dx.doi.org/10.5772/intechopen.96530*

as tracking and stopping transmission of the disease. Several hundreds of *P. falciparum* genomes have been generated in the past two decades. Genetic variation and function of various genes have improved our understanding of mutational changes, molecular structure, and evolutionary mechanisms in *Plasmodium*. There is an urgent need to retrieve utmost biological meaning from the available genomic data and translate such into tools that help resolve epidemiological challenges. This includes the identification of novel antigens for accurate and affordable diagnostic assays and vaccines, informative biomarkers that can distinguish different isolates and pinpoint the source of infections at fine geographical scale, and sensitive tool(s) for large-scale screening of asymptomatic infections in both high and low transmission areas. Future studies should examine how climate/environmental changes and selective pressure from interventions mediate genetic changes in the parasites, how host immune system responses to parasite changes, and how to uncover hidden parasite reservoirs and effectively control transmission.

#### **Acknowledgements**

the parasites [100]. Combining the anti-PfCyRPA c12 mAb with anti-RH5 BS1.2 mAbr will inhibit parasite growth *in vitro* from 21–31% to 59%. Thus, this antigen protein complex is expected to elicit a strong immune response against blood stage

TBV aims to prevent and kill the sexual stages of the *Plasmodium* parasites before transferring into the mosquito hosts. This vaccine offers protection against

*Pfs25* is an important glycophosphatidylinositol-linked protein expressed on the surface of ookinetes. It is found only within the *Anopheles* host and is approximately 25 kDa with 11 disulfie bonds [121]. The parasites require *Pfs25* to survive in the *Anopheles*'s midgut and develop into oocysts [122]. *Pfs25* is a conserve protein with low diversity. *Pfs25* vaccine was designed to elicit antibodies against the *Pfs25* antigen in humans and prohibit the development and transmission of gametocytes [98]. However, a recent study based on an adjuvant of *Pfs25* and a non-enveloped virus like protein (VLP) indicated weak IgG antibody responses in healthy individuals [123]. In another study, the antibody response of *Pfs25* proteins combined with four different adjuvants including alum, Toll-like receptor 4 (TLR-4) agonist glucopyranosal lipid A (GLA) plus alum, squalene–oil-in-water emulsion, and GLA plus squalene–oil-in-water emulsion were compared in mice. *Pfs25* combined with GLA plus squalene–oil-in-water emulsion was shown to induce the highest amounts of IgG antibodies [124]. Further studies should examine the formulation of this

*Pfs230* and *Pfs48/45*-based vaccines are antigens rich in cysteine produced by the

WHO aims to achieve malaria elimination in at least 35 countries, reduce incidence and mortality rates by 90%, and prevent resurgence in malaria-free countries by 2030. This ambitious goal has been challenged by the emergence and spread of antimalarial resistance, inaccurate diagnostic testing, asymptomatic transmission, and lack of effective vaccines [126]. Information of the *Plasmodium* genomes allow us to improve and reinvent tools/techniques for monitoring parasite changes as well

sexual stage gametocytes [97]. *Pfs48/45* can be found on the plasma membrane surface of both male and female gametocytes and are bounded to a GPI anchor that form a complex with *Pfs230* [125]. *Pfs230* is a 230 kDa protein that contains 14 6 cysteine rich domains [97]. *Pfs48/45*, on the other hand, contains three 6-cysteine rich domains [125]. The 6-cysteine rich domains of these antigens are essential for the formation of disulfide bonds on epitopes needed for antibody elicitation [125]. A recent study showed that mice injected with fragments of *Pfs48/45* and *Pfs230* prodomain produced higher levels of antibodies that induced complement fixation [125]. *Pfs230* and *Pfs48/45* combined with a FAB fragment of a monoclonal antibody 4F12 have been shown to further increase vaccine efficacy [97]. This vaccine is

currently in early clinical development phase [97, 125].

*P. falciparum* [120].

*5.3.1 Pfs25*

infection and transmission.

vaccine for better efficacy.

*5.3.2 Pfs230 and Pfs48/45*

**6. Conclusion**

**208**

**5.3 Transmission blocking vaccine (TBV)**

*Current Topics and Emerging Issues in Malaria Elimination*

We thank our colleagues at the University of North Carolina at Charlotte and the University of Ghana for the inspiration and discussion that lead to the conceptualization of this paper. We also thank the reviewers for their insightful comments.

## **Conflict of interest**

The authors declare no conflict of interest.

#### **Author contributions**

Conceptualization, C.C.D. and E.L.; resources, D.A.J. and E.L.; writing—original draft preparation, C.C.D., C.T.F., J.H., and E.L.; writing—review and editing, C.C. D., C.T.F., L.E.A, Y.A.A., D.A.J., and and E.L.; funding acquisition, E.L. All authors have read and agreed to the published version of the manuscript.

#### **Funding**

This research was funded by NIH/NIAID, grant number R15 AI138002.

#### **Author details**

Cheikh Cambel Dieng<sup>1</sup> \*, Colby T. Ford<sup>2</sup> , Jennifer Huynh<sup>1</sup> , Linda E. Amoah3,4, Yaw A. Afrane<sup>5</sup> , Daniel A. Janies<sup>2</sup> and Eugenia Lo<sup>1</sup> \*

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*DOI: http://dx.doi.org/10.5772/intechopen.96530*

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1 Department of Biological Sciences, University of North Carolina at Charlotte, Charlotte, USA

2 Department of Bioinformatics and Genomics, University of North Carolina at Charlotte, Charlotte, USA

3 Department of Immunology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana

4 West Africa Center for Cell Biology of Infectious Pathogens, University of Ghana, Accra, Ghana

5 Department of Medical Microbiology, University of Ghana Medical School, University of Ghana, Accra, Ghana

\*Address all correspondence to: cdieng@uncc.edu and eugenia.lo@uncc.edu

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Progress in Parasite Genomics and Its Application to Current Challenges in Malaria Control DOI: http://dx.doi.org/10.5772/intechopen.96530*

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**Author details**

Yaw A. Afrane<sup>5</sup>

Charlotte, USA

Accra, Ghana

**210**

Cheikh Cambel Dieng<sup>1</sup>

Charlotte, Charlotte, USA

University of Ghana, Accra, Ghana

University of Ghana, Accra, Ghana

provided the original work is properly cited.

\*, Colby T. Ford<sup>2</sup>

*Current Topics and Emerging Issues in Malaria Elimination*

, Daniel A. Janies<sup>2</sup> and Eugenia Lo<sup>1</sup>

1 Department of Biological Sciences, University of North Carolina at Charlotte,

2 Department of Bioinformatics and Genomics, University of North Carolina at

3 Department of Immunology, Noguchi Memorial Institute for Medical Research,

4 West Africa Center for Cell Biology of Infectious Pathogens, University of Ghana,

5 Department of Medical Microbiology, University of Ghana Medical School,

\*Address all correspondence to: cdieng@uncc.edu and eugenia.lo@uncc.edu

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Jennifer Huynh<sup>1</sup>

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*Current Topics and Emerging Issues in Malaria Elimination*

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[122] McLeod B, Miura K, Scally SW, Bosch A, Nguyen N, Shin H, et al. Potent antibody lineage against malaria

[123] Chichester JA, Green BJ, Jones RM, Shoji Y, Miura K, Long CA, et al. Safety

[124] Patra KP, Li F, Carter D, Gregory JA, Baga S, Reed SG, et al. Alga-

produced malaria transmission-blocking vaccine candidate Pfs25 formulated with

[125] Singh SK, Thrane S, Chourasia BK, Teelen K, Graumans W, Stoter R, et al.

a human use-compatible potent adjuvant induces high-affinity antibodies that block Plasmodium falciparum infection of mosquitoes. Infect Immun. 2015 May;83(5):1799–

transmission elicited by human vaccination with Pfs25. Nat Commun. 2019 09;10(1):4328. [PubMed Central: PMC6760140] [DOI:10.1038/s41467- 019-11980-6] [PubMed:31551421].

and immunogenicity of a plantproduced Pfs25 virus-like particle as a transmission blocking vaccine against malaria: A Phase 1 dose-escalation study in healthy adults. Vaccine. 2018 09;36

(39):5865–5871.

1808.

32355320].

309.

Hyg. 2017 Nov;97(5):1581–1592. [PubMed Central:PMC5817778] [DOI: 10.4269/ajtmh.17-0359] [PubMed:

[114] Morita M, Takashima E, Ito D, Miura K, Thongkukiatkul A, Diouf A, et al. Immunoscreening of *Plasmodium falciparum* proteins expressed in a wheat germ cell-free system reveals a novel malaria vaccine candidate. Sci Rep. 2017 04;7:46086. [PubMed Central:

PMC5380959] [DOI:10.1038/ srep46086] [PubMed:19874576].

platform for binding to human erythrocytes by Plasmodium

(37):25655–25669.

8910611].

2017;2.

6218–6227.

**220**

[115] Lin CS, Uboldi AD, Marapana D, Czabotar PE, Epp C, Bujard H, et al. The merozoite surface protein 1 complex is a

falciparum. J Biol Chem. 2014 Sep;289

[116] Wang YN, Lin M, Liang XY, Chen JT, Xie DD, Wang YL, et al. Natural selection and genetic diversity of domain I of *Plasmodium falciparum* apical membrane antigen-1 on Bioko Island. Malar J. 2019 Sep;18(1):317. [PubMed Central:PMC6751645] [DOI: 10.1186/s12936-019-2948-y] [PubMed:

[117] Srinivasan P, Baldeviano GC, Miura K, Diouf A, Ventocilla JA, Leiva KP, et al. A malaria vaccine protects Aotus monkeys against virulent Plasmodium falciparum infection. NPJ Vaccines.

[118] Sirima SB, Durier C, Kara L, Houard S, Gansane A, Loulergue P, et al.

Safety and immunogenicity of a recombinant *Plasmodium falciparum* AMA1-DiCo malaria vaccine adjuvanted with GLA-SE or Alhydrogel® in European and African adults: A phase 1a/1b, randomized, double-blind multicentre trial. Vaccine. 2017 10;35(45):

16111789].

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**Chapter 13**

Nigeria

**Abstract**

medicine vendors

**1. Introduction**

**223**

Using an Educational Training

Module to Increase Knowledge,

Attitudes and Practices of Malaria

among Medicine Vendors in Yobe,

*Yahaya Mohammed Katagum, Hayati Binti Kadir Shahar, Faisal Bin Ibrahim, Anisah Baharom and Rafee Baharudin*

This training module focuses on providing basic guidance on the current recommended approaches regarding malaria basic information, signs/symptoms, case detection, treatment, referral, and effective prevention strategies. The module can be used for in-service training programs on malaria or to assist in improving other health educator's work as well as serve as referral handbook for practicing health professionals. It can thus be used as a stand-alone training or together with modules dealing with other aspects of malaria control, prevention and elimination. The module uses a problem-solving approach to facilitate understanding and thereby motivate trainees on improved malaria case management. In essence, the training aims to generally improve the knowledge, attitudes and practices (KAP) of the most common handlers of malaria cases in this part of the world, the training module is then expected to improve services obtained by the majority of patients on malaria. On completion of training using this module, trainees will have acquired appreciable knowledge and skills on malaria basic-information, signs/symptoms, case detection/differentials, recommended drug treatment/appropriate dosing, indication for referral of complicated cases, effective prevention methods and the

**Keywords:** malaria KAP training, case detection, management and prevention,

Malaria remains a major global public health and development challenge over the years and in its World Malaria Report 2019, the World Health Organisation's (WHOs) global tally of malaria in 2018 alone was estimated at 228 million cases and 405,000 deaths [1]. Malaria is holo-endemic in many parts of sub-Saharan Africa thereby infecting citizens all year round and hence impacting the most devastation in this region. Worldwide, about 88 countries still remain at risk of malaria, with the types of species, severity and disease trends depending on the geographical location.

need to sensitise communities to stand up against malaria.

#### **Chapter 13**
