Introductory Chapter: Malaria in 2022 – Promises and Unmet Needs

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 (seeTable 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).

Plasmodium species	Incubation period	Periodicity of fever increase	Sign and Symptoms for severe disease
P. falciparum	9–14 days	3 days	Impaired consciousness: Glasgow Coma Scale score < 11;
			Multiple convulsion: > 2 seizures/days;
			Prostration: unable to sit, stand or walk alone;
			Significant bleeding: recurrent or prolonged bleeding from the nose, gum, venipuncture sites, hematemesis, or melena;
			Shock: circulatory collapse/shock.
P. vivax	12–17 days	3 days	Defined as falciparum malaria but w/o parasite density threshold.
P. ovale	16–18 days	3 days	Rarely occur.
			No parasite density threshold.
P. malariae	18–40 days	4 days	Rarely occur.
			No parasite density threshold.
P. knowlesi	9–12 days	2 days	Defined as for falciparum malaria except for parasite density (>100.000/mL) and for Jaundice and parasite density (>20.000/mL)

Table 1.

Characteristics of Plasmodium species infection and diagnosis criteria.

Modified by Daily et al. [5].

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 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.

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-malaria-transmission-cycle.

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].

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 low-density 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, 14, 15, 16, 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, 19, 20, 21, 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, 24, 25, 26].