Preface

Malaria is an ancient disease, and references to what was almost certainly malaria can be found in a Chinese document from about 2700 BCE, in Mesopotamian clay tablets from 2000 BCE, in Egyptian papyri from 1570 BCE and in Hindu texts as far back as the sixth century BCE. While these historical records should be regarded with caution, more recent observations are certainly stronger. The early Greeks, including Homer in about 850 BCE, Empedocles of Agrigentum in about 550 BCE and Hippocrates in about 400 BCE, were aware of the characteristic poor health, malarial fevers and enlarged spleens seen in people living in marshy places. For over 2500 years the idea that malarial fevers were caused by miasmas rising from swamps persisted, and it is widely held that the word malaria comes from the Italian malaria, meaning spoiled air, although this has been disputed. With the discovery of bacteria by Antonie van Leeuwenhoek in 1676, the recognition of microorganisms as causes of infectious diseases, and the development of the germ theory of infection by Louis Pasteur and Robert Koch in 1878–1879, the search for the cause of malaria intensified. Scientific studies only became possible after the discovery of the parasites themselves by Charles Louis Alphonse Laveran in 1880 and the identification of mosquitoes as the vectors, first for avian malaria by Ronald Ross in 1897 and then for human malaria by the Italian scientists Giovanni Battista Grassi, Amico Bignami, Giuseppe Bastianelli, Angelo Celli, Camillo Golgi and Ettore Marchiafava between 1898 and 1900.

Since then, progress has been made in malaria knowledge, prevention, and treatment. Quinine alone has probably saved millions of lives since its formal introduction in the 20th century. Nonetheless, in that time frame, malaria has claimed between 150 million and 300 million lives, accounting for 2 to 5 percent of all deaths. Although its chief sufferers today are the poor of Sub-Saharan Africa, Asia, the Amazon Basin, and other tropical regions, 40 percent of the world's population still lives in areas where malaria is transmitted (**Figure 1**).

This book presents a brief overview of the pathobiology of malaria and the current standards of diagnosis and treatments, and provides some insights into novel preventive and therapeutic approaches.

While the primary responsibility for malaria prevention and treatment lies with medicine and pharmacology, political as well as social initiatives are no less important. Most countries where malaria was endemic at the beginning of the 20th century are now totally malaria free. It is conceivable that investments in those areas where malaria is still deadly would lead not only to obvious global health benefits, but also to economic benefits, given that malaria, HIV and other infective diseases represent major obstacles to the development of such countries.

This book is intended for all health professionals, particularly those involved in THE diagnosis and treatment of tropical and infectious diseases, as well as medical students and researchers.

**Figure 1.**

*Malaria cases worldwide in 2020 (data from WHO). Nigeria (26.8%), the Democratic Republic of the Congo (12.0%), Uganda (5.4%), Mozambique (4.2%), Angola (3.4%) and Burkina Faso (3.4%) accounted for 55% of all cases.*

I am grateful to IntechOpen for the opportunity to edit this volume. I would like to thank Ms. Marica Novaković and Mr. Josip Knapic for their help and support, as well as all the contributors who have dedicated their time and effort to bringing this book to publication.

> **Pier Paolo Piccaluga, MD, Ph.D.** Professor, Biobank of Research and Institute of Hematology and Medical Oncology "Seràgnoli", IRCCS Azienda Ospedaliero Universitaria di Bologna S. Orsola-Malpighi, Department of Experimental, Diagnostic and Specialty Medicine, Bologna University School of Medicine, Bologna, Italy

> > **1**

Section 1

Introduction

Section 1 Introduction

#### **Chapter 1**

## Introductory Chapter: Malaria in 2022 – Promises and Unmet Needs

*Erica Diani, Davide Gibellini and Pier Paolo Piccaluga*

#### **1. Introduction**

Malaria represents one of the most ancient and diffuse infective illnesses in the World. It was first described in a Chinese paper, dated 2700 before Christ, in which a patient with recurrent fever was described. Many records on papyri and clay tablets report similar cases, but the definition of causative agent of malaria had to wait for the germ theory and the first discovery of *Plasmodium* in human blood sample [1, 2]. After almost 5000 years, malaria continues to be frightening: in 2020, the WHO estimated 241 million cases and 627,000 deaths in the 85 countries in which malaria is endemic (WHO), and about of 75% of infected patients are children. In recent decades, various programs have been implemented in order to contain and reduce the transmission of malaria through prevention, diagnosis, and surveillance strategies. Unfortunately, the SARS-CoV-2 pandemic has caused an increase in the number of cases and deaths due to the interruption of malaria prevention and a sort of black out in the surveillance and case reports. Now, after 2 years of SARS-CoV-2 pandemic, the number of malaria cases and death has not been even updated; the last officially released data were in 2019, but it is absolutely necessary to maintain open the attention on this plague [3].

#### **2. The biological cycle of** *Plasmodia*

Malaria is a life-threatening disease characterized by recurrent periodically fever accompanied by nausea, vomiting and abdominal discomfort, fatigue, and headaches [4]. Infection is caused by five protozoan parasites of the genus *Plasmodium*: *P. falciparum, P. vivax, P. ovale, P. malariae,* and *P. knowlesi,* which are characterized by different periodicity and different severity of illness (see **Table 1**). In particular, *P. falciparum* is responsible for malignant tertian, which, in the more severe cases, can lead to the death of patients in 24 hours after the symptoms appear (WHO).

Transmission occurs through an arthropod vector, which can acquire the *Plasmodium* through the bite of an infected person. *Plasmodium* maturation proceeds in the Anopheles stomach and midgut, and the sporozoites (infective form) are released in another person by mosquito bite, starting new infection cycle [6, 7]. Rarely, the transmission can occur among persons through blood transfusion or organ transplants.

The biological cycle of *Plasmodia* takes place in two obligated hosts (**Figure 1**): a vertebrate and a female of mosquito Anopheles. In brief, the infection of a female


#### **Table 1.**

*Characteristics of Plasmodium species infection and diagnosis criteria.*

Anopheles occurs after a blood meal carried out on an infected human subject carrying the gametocytes, the sexual forms of the parasite, which are the only ones that can proceed with the development in the Anopheles body. The sporozoites are the final stage of development cycle and are also the human infective form of *Plasmodium*. At the end of the development cycle, the sporozoites, the forms of *Plasmodium* infecting humans, migrate into the salivary glands of the mosquito, from where they will be inoculated into another human subject through the bite. The development of the *Plasmodium* continues also inside the human host. Sporozoites can reach the liver, establishing a silent infection in the hepatocytes and undergoing a strong proliferation with the formation of the schizont within which maturation takes place in the form of merozoites. When the merozoites are mature, the schizont ruptures release them into the bloodstream where they invade the erythrocytes causing disease. Within the erythrocytes, the merozoites pass through another developmental stage, starting from ring form, to trophozoites, and ending to multinucleated schizonts (erythrocytic stage). This final step in the red blood can undergo a cycle (the intraerythrocytic developmental cycle, IDC) activated by the rupture of the schizont continuing the infectious phase where they reproduce again by schizogony, giving rise to new generations of parasites every 48 (tertian) or 72 (quartana) hours.

Some merozoites can further differentiate into female and male gametocytes, which, being present in the bloodstream of the infected patient for several weeks, can be ingested during a mosquito's meal, thus initiating a new transmission. Only for *P. vivax* and *P. ovale*, the liver stage infection is also characterized by a dormant phase also called hypnozoite, which causes a prolonged infection [8].

*Introductory Chapter: Malaria in 2022 – Promises and Unmet Needs DOI: http://dx.doi.org/10.5772/intechopen.109328*

 **Figure 1.**

 *Biological cycle of Plasmodium in human host. Reprinted from "Malaria Transmission Cycle", by BioRender, May 2019— https://app.biorender.com/biorender-templates/t-5e629f969501410088a0156b-malariatransmission-cycle .* 

#### **3. Malaria diagnosis**

 The first steps to try to reduce malaria cases and death are obvious: protective clothing, mosquito repellants, bed nets, screened accommodations, chemoprophylaxis (only for travelers in the endemic areas), avoid visiting friends infected, which is the cause of about 50% of new infections [ 3 ].

 In October 2021, WHO approved the use of the malaria vaccine, RTS,S/ASO1 (GSK Belgium), in children from 5 months of age. This vaccine is based on a recombinant subunit protein of P. falciparum that is expressed during sporozoite stage. This new strategy is leading to reduced new malaria cases worldwide [ 9 ]. Recent study on the vaccination campaign impact reports that in a population of children from 5 months age, who received all four doses of malaria vaccine, in a follow-up time of 4 years, they observed a reduction in both clinical and severe malaria cases of 39% and 29%, respectively [ 10 ].

 The second important step to eradicate and contain malaria contagion in endemic areas (including cases derived from traveling) is a correct and tempestive diagnosis of malaria with specific indication of which species of malaria is or are present in the sample, in order to use the most appropriate drug treatment.

 Following the indication of WHO and of the British Society of Hematology [ 3 , 11 ], the diagnostic assays for malaria are: microscopic examination in thin and thick film, Rapid Diagnostic Test (RDT) based on antigen, Indirect Fluorescent Antibody Test (IFA test), and also, recently introduced and accepted, the nucleic-acid detection method by PCR or LAMP technologies.

#### **3.1 Microscopy**

It is currently the gold standard to diagnose malaria. This technique requires experienced hematologists and technicians to prepare and observe blood smears, discriminate different *Plasmodium* species, and correctly quantitate the parasite density in the specimen. When *P. falciparum* or *P. knowlesi* are detected, it is very important to determine the parasite percentage because the species and the parasitemia level may affect the treatment choice.

In brief, for each patient, two thick and two thin films of blood smears should be prepared, starting from a venipuncture performed maximum 2–4 h earlier, in order to avoid morphological alteration due to EDTA storage. Double preparation is needed for independent analyses of the slides by two specialists and for greater control and precision in the parasite count.

Thick and thin films have two different aims: detection of parasite and identification of species, respectively. In addition, for the species indicated in thin film, it can be possible to perform the parasite count, taking into account only the asexual stage of the parasites (ring and merozoite). The films are colored by Giemsa stain at pH 7.2. For severe illness, it can be possible to perform a modified field stain in order to detect more rapidly *P. falciparum* and start as soon as possible the adequate treatment.

The great advantage of this technique relies in its relative simplicity; in fact, it can be performed in any laboratory that performs hematology tests, requiring no additional equipment. In addition, microscopy can provide three important data to start the patient treatment: presence of *Plasmodium*, its specie, and count. The main disadvantage is that morphological analysis requires experienced staff.

#### **3.2 Rapid diagnostic test**

Rapid diagnostic test is a faster alternative to microscopy detection. This immunochromatographic test is performed starting from some small drops of blood, and in about 15 minutes, the physician can visualize the presence of specific bands in the window of the test card. This immunochromatographic test, based on antigen or antibody, makes it possible to identify four out of five *Plasmodium* species, *P. knowlesi* being excluded.

This approach allows a faster but less sensitive diagnosis and might be not sufficient. The main advantage is that it does not require expert personnel and can even be self-performed, facilitating diagnosis where the doctor cannot reach the patients. On the other hand, as a limitation, we can only obtain information about the presence of one or more species of *Plasmodium*, but the amount of parasites is unveiled.

#### **3.3 Indirect fluorescent antibody test**

Indirect Fluorescent Antibody (IFA) test is based on the detection of antibodies in the patient serum. Due to the long time needed to carry out the procedure, it cannot be adopted as a routine test for malaria detection. However, IFA test is useful to screen the blood donors in malaria cases suspected to be transmitted by hemo-transfusions, when a donor is negative at microscopy test. The use of specific antibody allows to detect *Plasmodium* also in infected patient with very low parasitemia. In addition, IFA test is a good tool for the test of patient with chronic or repeated malaria infection and for patient whose diagnosis is unsure after starting drug therapy.

IFA test is available for all *Plasmodium* with exception of *P. knowlesi*, due to the availability of specific antibodies.

Another serology test employed is an immune-enzymatic assay, used principally for blood donors screening. The limit of this test is its sensitivity due to the possibility to detect only *P. falciparum vs "non-falciparum spp."*

#### **3.4 Nucleic acid detection methods**

To date, only a few referral laboratories use molecular methods to detect, define, and quantify *Plasmodium* infections, while these tests are principally used for research and epidemiologic scopes [12]. Only for a suspect of *P. knowlesi* infection, polymerase chain reaction (PCR) is becoming a standard to confirm the diagnosis before treatment initiation.

PCR-based techniques could be useful to detect malaria in case of infection at lowdensity parasitemia, which is difficult to detect also at microscopy or in the absence of an expert technician. Of note, the specific oligonucleotides used for the amplification allow, at the same time, detection, species definition and quantitation, even for low amounts of *Plasmodia* [13–17]. Prospectively, PCR-based tools are expected to be the gold standard in malaria diagnosis, as already happened in most infectious diseases. In fact, PCR is currently cheap, easy to perform even in low resources settings, fast, and absolutely accurate.

Loop-mediated isothermal amplification test (LMPA) can detect parasite DNA in a simpler way with respect to PCR [18–22]. The advantages of LMPA tests, compared with PCR, are that thermocyclers are not required. Conversely, sensitivity and specificity are variable, making this test not always reliable [23–26].

#### **Author details**

Erica Diani1 , Davide Gibellini1 and Pier Paolo Piccaluga2,3,4\*

1 Microbiology Section, Department of Diagnostic and Public Health, Verona University, Italy

2 Biobank of Research and Institute of Hematology and Medical Oncology Seràgnoli, IRCCS Azienda Ospedaliera-Universitaria S. Orsola-Malpighi, Bologna, Italy

3 Department of Experimental, Diagnostic, and Experimental Medicine, Bologna University School of Medicine, Bologna, Italy

4 Jomo Kenyatta University of Agriculture and Technology and University of Nairobi, Kenya

\*Address all correspondence to: pierpaolo.piccaluga@unibo.it

© 2022 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.

#### **References**

[1] Cox FE. History of human parasitology. Clinical Microbiology Reviews. 2002;**15**(4):595-612. DOI: 10.1128/CMR.15.4.595-612.2002. Erratum in: Clinical Microbiology Reviews. 2003 Jan;**16**(1):174. PMID: 12364371; PMCID: PMC126866

[2] Cox FE. History of the discovery of the malaria parasites and their vectors. Parasites & Vectors. 2010;**3**(1):5

[3] World Health Organization. WHO Guidelines for Malaria. Geneva: World Health Organization; 2022. Available from: http://apps.who.int/bookorders

[4] White NJ, Pukrittayakamee S, Hien TT, Faiz MA, Mokuolu OA, Dondorp AM. Malaria. The Lancet. 2014;**383**(9918):723-735

[5] Daily JP, Minuti A, Khan N. Diagnosis, treatment, and prevention of malaria in the US. Journal of the American Medical Association. 2022;**328**(5):460

[6] Aly ASI, Vaughan AM, Kappe SHI. Malaria parasite development in the mosquito and infection of the mammalian host. Annual Review of Microbiology. 2009;**63**(1):195-221

[7] Frischknecht F, Matuschewski K. *Plasmodium* sporozoite biology. Cold Spring Harbor Perspectives in Medicine. 2017;**7**(5):a025478

[8] Josling GA, Williamson KC, Llinás M. Regulation of sexual commitment and Gametocytogenesis in malaria parasites. Annual Review of Microbiology. 2018;**72**(1):501-519

[9] World Health Organization. WHO Recommends Groundbreaking Malaria Vaccine for Children at Risk. 2021

[10] Praet N, Asante KP, Bozonnat MC, Akité EJ, Ansah PO, Baril L, et al. Assessing the safety, impact and effectiveness of RTS,S/AS01E malaria vaccine following its introduction in three sub-Saharan African countries: Methodological approaches and study set-up. Malaria Journal. 2022;**21**(1):132

[11] Rogers CL, Bain BJ, Garg M, Fernandes S, Mooney C, Chiodini PL. British Society for Haematology guidelines for the laboratory diagnosis of malaria. British Journal of Haematology. 2022;**197**(3):271-282

[12] Proux S, Suwanarusk R, Barends M, et al. Considerations on the use of nucleic acid-based amplification for malaria parasite detection. Malaria Journal. 2011;**10**:323. DOI: 10.1186/1475-2875-10-323

[13] Vafa Homann M, Emami SN, Yman V, Stenström C, Sondén K, Ramström H, et al. Detection of malaria parasites after treatment in travelers: A 12-months longitudinal study and statistical modelling analysis. eBioMedicine. 2017;**25**:66-72

[14] Steenkeste N, Incardona S, Chy S, Duval L, Ekala MT, Lim P, et al. Towards high-throughput molecular detection of *Plasmodium*: New approaches and molecular markers. Malaria Journal. 2009;**8**(1):86

[15] Okell LC, Ghani AC, Lyons E, Drakeley CJ. Submicroscopic infection in *Plasmodium falciparum* – endemic populations: A systematic review and meta-analysis. The Journal of Infectious Diseases. 2009;**200**(10):1509-1517

[16] Giha H, Hviid L, Richardson W, Elhassan IM, Arnot DE, Theander TG, *Introductory Chapter: Malaria in 2022 – Promises and Unmet Needs DOI: http://dx.doi.org/10.5772/intechopen.109328*

et al. Detection of very low level *Plasmodium falciparum* infections using the nested polymerase chain reaction and a reassessment of the epidemiology of unstable malaria in Sudan. The American Journal of Tropical Medicine and Hygiene. 1996;**54**(4):325-331

[17] Vannachone B, Kobayashi J, Toma H, Inthakone S, Manivong K, Nambanya S, et al. A field study on malaria prevalence in southeastern Laos by polymerase chain reaction assay. The American Journal of Tropical Medicine and Hygiene. 2001;**64**(5):257-261

[18] Notomi T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research. 2000;**28**(12):63e-663e

[19] Poon LL, Wong BW, Ma EH, Chan KH, Chow LM, Abeyewickreme W, et al. Sensitive and inexpensive molecular test for falciparum malaria: Detecting *Plasmodium falciparum* DNA directly from heat-treated blood by loopmediated isothermal amplification. Clinical Chemistry. 2006;**52**(2):303-306

[20] Hopkins H, González IJ, Polley SD, Angutoko P, Ategeka J, Asiimwe C, et al. Highly sensitive detection of malaria parasitemia in a malaria-endemic setting: Performance of a new loop-mediated isothermal amplification kit in a remote clinic in Uganda. The Journal of Infectious Diseases. 2013;**208**(4):645-652

[21] Patel JC, Lucchi NW, Srivastava P, Lin JT, Sug-aram R, Aruncharus S, et al. Field evaluation of a real-time fluorescence loop-mediated isothermal amplification assay, RealAmp, for the diagnosis of malaria in Thailand and India. Journal of Infectious Diseases. 2014;**210**(8):1180-1187

[22] Perera RS, Ding XC, Tully F, Oliver J, Bright N, Bell D, et al. Development and

clinical performance of high throughput loop-mediated isothermal amplification for detection of malaria. PLoS One. 2017;**12**(2):e0171126

[23] Thekisoe O, Pöschl B, Chutipongvivate S, Panagiotis K, Waneesorn J. Comparative diagnosis of malaria infections by microscopy, nested PCR, and LAMP in Northern Thailand. The American Journal of Tropical

[24] Piera KA, Aziz A, William T, Bell D, González IJ, Barber BE, et al. Detection of *Plasmodium knowlesi*, *Plasmodium falciparum* and *Plasmodium vivax* using loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia. Malaria Journal. 2017;**16**(1):29

Medicine and Hygiene. 2010;**83**(1):56-60

[25] Katrak S, Murphy M, Nayebare P, Rek J, Smith M, Arinaitwe E, et al. Performance of loop-mediated isothermal amplification for the identification of submicroscopic *Plasmodium falciparum* infection in Uganda. The American Journal of Tropical Medicine and Hygiene. 2017;**97**(6):1777-1781

[26] Morris U, Aydin-Schmidt B. Performance and application of commercially available loop-mediated isothermal amplification (LAMP) kits in malaria endemic and non-endemic settings. Diagnostics. 2021;**11**(2):336
