Malaria: biochemical, physiological, diagnostic, and therapeutic updates

Hypoglycemia induced by malarial infection

The disruption of glucose metabolism in malaria chiefly results from the sequestration of intraerythrocytic stages in the arteries of the liver in severe P. falciparum infection (Sengupta et al., 2020; Olszewski et al., 2009; Tian et al., 2022; Glaharn et al., 2018). Other mechanisms involve the inhibition of ATP-sensitive potassium (K_ATP) channels, which modulate the pancreatic beta-cell membrane’s permeability to potassium. This results in marked calcium influx and insulin release and reduction of the blood glucose level (Onyesom & Agho, 2011). In summary, one or more of the following can also contribute to hypoglycemia in malaria:

1. The malaria parasite consumes the host’s circulating glucose to meet its energy requirements: The host’s glucose supply is a major source of support for the parasites during the asexual phases, which they use to produce ATP through the process of glycolysis (Sengupta et al., 2020).

2. The metabolic modifications brought on by parasites that prevent gluconeogenesis: Infection with P. falciparum causes the release of cytokines such as tumor necrosis factor (TNF), interleukin (IL)-1, and 6, which block the activity of phosphoenolpyruvate carboxykinase, a crucial enzyme in the gluconeogenetic pathway (Mavondo et al., 2019).

3. Malaria-related increases in IL-1 and IL-6 release encourage islet cell hyperplasia resulting in an unregulated insulin response and subsequent reduction of the blood glucose level (Mavondo et al., 2019).

4. Erythrocytes can more easily absorb glucose when it is transported across the plasma membrane by GLUT1. The merozoite invasion results in insertion of numerous transmembrane proteins that lead to dramatic change in the plasma membranes of IEs (Autino et al., 2012).

5. Additional factors like malaria-related loss of appetite contribute to the downregulation of 5′ AMP-activated protein kinase (AMPK) gene expression, and reduced energy intake (Apoorv, Karthik & Babu, 2018). Moreover, the parasite modulated the enzyme 5′ AMP-activated protein kinase (AMPK) and lowered AMPK phosphorylation following infection thus influencing the energetic metabolism of the cell.

Dyslipidemia induced by malarial infection

Dyslipidemia in patients with malaria is implicated chiefly by the parasite. Plasmodium spp. depend on the host for some of the lipids necessary for their growth and development because some of their important lipids cannot be produced via intraerythrocytic biosynthesis (Okagu et al., 2022). By enhancing lipid absorption, de novo synthesis, lowering cellular uptake, and/or other strategies, the parasite manipulates the host’s circulating lipid levels to maximize the availability of these vital lipids. The results of a study showed raised TAG levels in the serum, white adipose, and liver, as well as greater free fatty acids and cholesterol in the hepatic tissues of chabaudi rodent malaria (Autino et al., 2012). Also, Kluck et al. (2019) deduced that P. chabaudi infection changes the hepatic mRNA and expression of key enzymes and transcription factors determent in the metabolism of lipids, thus resulting in a lipogenic state. However, variances in the modification of lipid levels in malaria appear to be affected by the severity of the illness and the species of Plasmodium parasite. For instance, P. vivax-infected patients were observed to have lower TC, LDL, and HDL levels while having higher TAG levels when compared to normal/apparently healthy individuals (Mesquita et al., 2016). Falciparum malaria has also been associated with decreased levels of HDL, LDL, and total cholesterol as well as increased or unaltered levels of TAGs and very low-density lipoproteins (VLDL) (Kullu et al., 2018). Of note, following antimalarial medication therapy, these changes in lipid levels normalized along with a significant decrease in parasitemia and the eradication of clinical symptoms of malaria. Mice infected with P. berghei NK-65 in experimental rodent malaria had higher serum lipid profile levels than control mice (Enechi, Okagu & Ezumezu, 2021).

Kidney functions.

Falciparum and Plasmodium malariae infections are associated with renal manifestations that can produce an immune complex-mediated glomerular disease-causing nephrotic syndrome (Naqvi, 2021). P. falciparum infection is associated with more renal tubular changes than glomerular changes, and acute renal failure (ARF) is a possible complication (Lendongo Wombo et al., 2023). Oliguria, high serum creatinine, and blood urea nitrogen are useful to diagnose ARF. The adverse effects of the malaria parasite on the kidney can cause hypernatremia, hyperkalemia, high blood urea, low urine specific gravity, and metabolic acidosis (Enechi et al., 2021).

Severe malaria infection is associated with increased urea and creatinine compared to mild infection. The renal ischemia caused by the sequestration of the parasite into the microvasculature bed of the kidney could be the cause of high urea and creatinine in severe malaria. It was observed that serum protein decreased markedly in severe malaria infection, which is thought to be due to the influence of the parasite on protein synthesis (Ozojiofor et al., 2020).

Malaria patients showed a decrease in electrolyte levels. The decrease in Na⁺, Cl⁻, and HCO₃ levels reflects the level of renal dysfunction, which is proportional to the severity of malaria infection. Low Na⁺ levels are explained by their loss in urine and sweat to compensate for the high lactase and urea levels of plasmodium falciparum-infected patients (Uwah et al., 2021). Meanwhile, K⁺ levels are high in severe infections, with a possible risk of developing metabolic acidosis. Malaria infection causes impaired glomerular filtration, which results in a decrease in Na+ available in the renal tubule for K ⁺ exchange. The resulting imbalance in hydrogen ions causes K+ retention, which can explain the high K⁺ serum levels (Enechi et al., 2021) (Fig. 1A).

Limitations of biochemical and physiological indices

To our understanding, the range of functional reference of any parameter is a resultant interaction of multiple physiological components. In malaria infection, another pathological component becomes added to the stream of biochemical reactions. This might highlight the important role exerted by laboratory professionals to use literature, interrelate data, and facilitate comprehension and prediction of diagnosis e.g., in patients with hypoglycemia and dyslipidemia and who have a travel history to malaria-endemic areas. Another issue, in case clinicians are relying on the physiological and biochemical indices to predict a diagnosis of malaria, it is important to assess sensitivity, specificity, and the rate of device failure to reduce rates of misdiagnosis. More studies for the comparative validity of physiological and biochemical indices for differential diagnosis of malaria infection from other metabolic conditions are still needed.

Rapid diagnostic tests or immunochromatographic tests

This type of device is considered a widespread point-of-care test (POCT) to diagnose malaria. Rapid diagnostic tests (RDTs) is a lateral flow immunoassay technique that detects the presence of biomarkers specific to the Plasmodium parasite. The technique is a single-step test with the advantage of being cheap and fast. However, the affordability and accessibility RDTs do not define them as distinctive malaria diagnostic factors (Wittenauer, Nowak & Luter, 2022). This device is comprised of a nitrocellulose membrane netted with antibodies and antibodies against the target antigen to detect several biomarkers.

Commercially, the available RDTs biomarkers involve P. falciparum histidine175 rich protein-II (Pf-HRP-II), which is water soluble and is expressed on the surface of the RBCs. Also, plasmodial aldolase (pALD), as well as plasmodial lactate dehydrogenase (pLDH), are parasite-specific glycolytic enzymes recognized in all plasmodium species. Yet, several drawbacks have been determined for RDTs. For instance, Pf-HRP-II is the most principal target to detect the parasite; yet, it is exclusively produced by P. falciparum and thus not sensitive for other species, has a detection limit >40–100 parasites per µL, persists as positive for up to 31 days post-treatment, reported for cross-reactivity either with other plasmodium species or autoantibodies; besides it cannot detect viability of the parasite. Regarding HRPII, it is released during schizogony into the bloodstream and remains positive for several weeks, even after the clearance of infection, shows a higher detection limit of parasites/µL, and is inefficient in differentiating parasite species (Grandesso et al., 2016). The pALD exhibits comparable drawbacks similar to HRPII, yet, its positive result defines the viability of the parasite.

The limitation of conventionally used RDT also involves the reduction in peripheral parasitemia owing to Plasmodium sequestration. Also, PfHRP2 antigen are also useful in Africa, India, Asia, middle east, Amazon regions in addition to sub-Saharan countries and South America, since some isolates lacking this antigen have been found from these areas. The the presence of Pf-HRP-II/III deletions in the parasites reinforces the necessity for novel diagnostic techniques for systematic surveillance in endemic regions (Berhane et al., 2018).

Biosensors as a recent “point-of-care”

Firstly, what are the biosensors? Biosensors are detector devices composed of immobilized bio-receptors (bio-recognition element) and a transducer. The bio-receptor might be an antibody, enzyme, DNA, or microorganism that detects and bind with the target analyte, and the produced changes in the physicochemical characteristics (e.g., electrochemical, magnetic, optical, and piezoelectric acaustic) are transformed into a quantitative or semi-quantitative electrical signal that can be measured by a transducer as shown in Fig. 2 (Ding, Srinivasan & Tung, 2015). Such devices were designated to explore for analytes like hemozoin and several other enzymatic markers e.g., LDH, glutamate dehydrogenase (GDH), merozoite surface protein 3 (MSP-3), and ALD in enzyme-based biosensors (Dutta, 2020).

Types of biosensors

SPR biosensors.

These sensors detect the reaction between the bio-receptors and the target analyte through the alteration in the refractive index of the plasma resonance substrate, the SPR angle, and the intensity of light reflectance. These types of biosensors are label-free, have high sensitivity and resolution, permit real-time evaluation, and possess a high signal-to-noise ratio; hence can be considered suitable for point-of-care applications of malaria disease. However, SPR bio-sensors are motion-sensitive and rely on the quality of the light indicator. They entail the adjustment of light distance and angle and are affected by the molecular size and concentration of the analyte. Therefore, full automation is necessary to avoid extensive calibration and light intrusions (Ragavan et al., 2018) as in (Fig. 3A).

There are several models for this type; for example, Chaudhary, Kumar & Kumar (2021) attempted to improve the SPR using two sheets of air holes in the form of a hexagonal lattice (photonic crystal fiber) and a tinny layer of gold (Au). Evaluation of this model showed a recognizable shift in the SPR resonance wavelength ( λ) and refractive index with malaria-infected red blood cells (RBCs) compared with normal RBCs. In addition, it was useful to discriminate early infections and the different stages of the parasite; for instance, ring stage at λ = 13,714.29 nm/RIU, trophozoite at λ = 9,789.47 nm/RIU, and schizont at λ = 8,068.97 nm/RIU (Chaudhary, Kumar & Kumar, 2021).

The lab-on-a-chip and micro-device technology

The novel microdevices, especially lab-on-a-chip devices, have been considered a potential portable point of care for the diagnosis of malaria. This technology relies on the concentration of RBCs through their margination using micro-channels for more specific and sensitive detection. The design of the micro-channels simulates blood capillaries with a caliber of less than 300 µm. It is well known that RBCs are more deformable and smaller than white blood cells (WBCs). Therefore, RBCs under normal conditions flow faster than WBCs along the axial center of the blood capillaries; whereas, WBCs marginate to the endothelium of the blood capillaries. In malaria infection, the stiffer infected RBCs act similarly to WBCs and show cytoadherence to the endothelium (Giacometti et al., 2021).

In the fabricated micro-channel, P. falciparum-infected RBCs align along the sidewalls “via margination”; thereafter, infected RBCs can be removed and separated using a three-outlet system as shown in Fig. 4A (Kolluri, Klapperich & Cabodi, 2018). Another model was invented that relied on the magnetic properties of the hemozoin to fasten the margination of the infected RBCs in a magnet-based microfluidic device. The device is coupled to a nickel wire that attracts infected RBCs and a permanent magnet to create an external field (∼0.6 T). Thus, allowing the separation of malaria-infected RBCs as in Fig. 4B (Nam et al., 2019).

Multiplex human malaria array.

A new sensitive generation of rapid diagnostics for the quantification of malaria antigens may facilitate the screening of asymptomatic malaria. The Q-Plex™ Human Malaria Array (Quansys Biosciences, Logan, UT, USA) has been evaluated to quantify the antigens specific for Plasmodium spp. e.g., HRP-2, Pf-LDH, Pv-LDH, and Pan-LDH. The new kit showed 99.5 improved specificity whereas sensitivities against PCR were 92.7%, 71.5%, 46.1%, and 83.8% for HRP2, Pf-LDH, Pv-LDH, and Pan LDH, respectively (Jang et al., 2020).

Chemical falcipain inhibitors

Falcipains are cysteine protease enzymes related to the papain family. The Papain family of proteins has a wide range of aminopeptidases dipeptidyl peptidases, endopeptidases, and enzymes with both exo- and endopeptidase action. Falcipain-2 and -3 are key protease enzymes produced by the P. falciparum parasites and are involved in the hydrolysis of hemoglobin and the production of free amino acids. Falcipain-2 is convoluted in slicing band 4.1 protein and ankirin involved in the cytoskeleton of the red blood cell. Interestingly, other Plasmodium species express proteases homologs to falcipain-1, -2 and-3 proteases. Therefore, falcipain inhibitors are regarded as promising agents against other parasite species (Rosenthal, 2020; Ettari et al., 2021). Examples involve (1) quinoline-4-carboxamide derivative. This compound was developed using molecular hybridization. The efficacy of quinoline-4-carboxamides involves the inhibition of falcipain-2; besides altering the food-vacuole and morphology of the parasite (Singh et al., 2021). (2) The (E)-chalcone inhibitor. Chalcones are organic compounds with antimalarial and antioxidant activities. E-chalcone 48 was found to bind only to the falcipain-2-substrate binding cleft; thus, achieving high specificity (Machin, Kantsadi & Vakonakis, 2019). (3) Tetracycline. Tetracycline is a broad-spectrum antibiotic; where its derivatives were found to inhibit Falcipain-2 by interacting with its distal allosteric site (Hernández González et al., 2021).

Plant extracts

Medicinal plants proved to have impressive anti-plasmodial activities. Interestingly, studies afford the scientific basis of plant extracts and their adventitious usage in folkloric herbal medicine. Herein, we presented some examples (Laryea & Borquaye, 2019). Examples involve (1) Buchholzia coriacea Engl. Seeds. In a murine study, a flavonoid-rich extract of B. coriacea seeds (FEBCS) had a dose-dependent chemo-suppression; yet its efficacy was lower than combisunate. FEBCS improved the hematological and biochemical parameters to near-normal levels with no morphological or behavioral sign toxicity (Enechi et al., 2021). (2) Gymnema inodorum Leaf. In murine models, this plant extract exhibited protective effects against the biochemical changes due to Plasmodium berghei e.g., hypoglycemia, dyslipidemia, and acute liver and Kidney injury (Boonyapranai et al., 2021a); in addition, alterations in hematological (Ounjaijean et al., 2021) and blood coagulation parameters were normalized (Boonyapranai et al., 2022b). (3) Ethanol extract of the stem bark of Anogeissus leiocarpus (EESAL). Treatment of malaria-infected murine models with EESAL showed improved hematological parameters, restored biochemical indicators for lipid peroxidation and liver functions, and exerted antioxidant effects. Besides, EESAL was tolerable per oral up to 5,000 mg/kg body weight (Dahiru, Badgal & Neksumi, 2023). (4) Ethanol extract of Triumfetta cordifolia. A Nigerian study revealed the efficient anti-plasmodial activity of T. cordifolia; yet, its mechanism of action is still not recognized (Ezenyi et al., 2020). (5) Bidens pilosa, Syzygium guineense and Parinari congensis extracts. In mammalian cell lines, these extracts exhibited very feeble to no cytotoxicity. Yet, it showed high selectivity for Plasmodium parasites. Extracts from Syzygium guineense and Parinari congensis were the most toxic against the parasite (Laryea & Borquaye, 2019).

Nano-medicine

Why is nano-medicine regarded as the new era in malaria treatment? Plasmodium parasites evade the host immune system within the RBCs. Thus, targeting the parasite necessitates the ability of the drug to cross membranous barriers of the RBCs and the parasitophorous vacuole and plasma membrane of the Plasmodium parasite. Thereafter, the drug, in an optimum dose, should reach a definite target e.g., a food vacuole or an apicoplast membrane of the parasite (Anamika et al., 2020). Yet, nanocarriers are less than 1,000 nm with a high surface area: volume ratio; thus, allowing for improving the area under curve (AUC curve) and efficacy of the drugs with lower doses and frequencies of administration, improved half-life time, and water solubility (Rathee et al., 2015; Patra et al., 2018).

Overall, antimalarial drugs can approach their targets either through the lipid membrane of the RBCs by diffusion or through interacting with the protein component of the erythrocyte membrane (receptors and transporters) by carrier-mediated transportation. Carrier-mediated transportation is either passive or active (Clemons et al., 2018). In the case of passive carrier-mediated transportation energy is not required; hence considered facilitated diffusion. This type is attained by the conventional nanocarriers (the liposomes, the polymeric nanoparticles, and the long-circulating polyethylene glycol nanocarriers (PEGylated)). Nanocarriers, which are less than 80 nm in diameter, leak through the porous membrane of the infected RBCs. In this case, the nano-carrier bypasses the cytosol of the RBCs and sinks directly to the parasite through the tubovesicular membrane (TVM). TVM is an alteration in the architecture of the RBCs wherein a network is synthesized by the parasite to connect the membrane of the RBCs with the membrane of the parasitophorous vacuole (that surrounds the parasite inside the RBCs). This TVM facilitates Plasmodium-derived proteins transport, nutrition uptake, and elimination of waste products outside the cell; thus, allowing the thriving of the parasite (Rai et al., 2017). For example, human serum albumin (HSA) has been recognized as a potential drug-delivery vehicle for artemether (ATM) in models infected with P. berghei species. These nanoparticles showed two-fold higher peaks in the drug concentrations inside the RBCs. Overall, HSA is non-toxic and non-immunogenic (Kaur et al., 2021). In addition, HSA is an endogenous transport protein with several drug binding sites; thus, acting as a useful drug carrier to improve stability and half-life time of the loaded drugs or perform as a targeting agent. HSA has high specificity and uptake by the parasitized RBCs (Tahir, Malhotra & Chauhan, 2003). In 2019, Esu et al. (2019) speculated that HSA can intensely bind to ATM and its byproducts by thiol and amino groups, and thus can be used in drug-resistant.

On the contrary, active drug targeting requires energy consumption and surface activation of the nanocarriers by using specific ligands that bind to receptors on the membrane of the infected RBCs. These ligands might be in the form of antibodies, carbohydrates, peptides, or proteins. In this type, the drug is delivered to the parasite through the cytosol of the RBCs (Anamika et al., 2020). For instance, pyronaridine and atovaquone can be formulated as immunoliposomes and interact with the glycophorin-A receptor on the RBCs through anti-glycophorin A-antibodies (Biosca et al., 2019). Artemisinin can be loaded on nanostructured lipid carriers and react with the transferrin receptor on the brain cells (in cerebral malaria) through transferrin ligands (Emami, Yousefian & Sadeghi, 2018). Recently, the emergence of the polymeric wall in the encapsulation of lipophilic (oil) drugs has been introduced. These Nanocapsules have high entrapment efficacies and low toxicity and polymer content. In addition, it increases the solubility of the compounds and evades drug inactivation in the gut (Rajabi & Mousa, 2016). In a prior study, the poly (butyl methacrylate-co-morpholino ethyl sulfobetaine methacrylate)-based nanoparticles were shown to specifically target P. falciparum-infected RBCs compared with the healthy RBCs by a ratio 74.8%: 0.8% (Biosca et al., 2021).

The combination therapy

To compete challenges in malaria treatment combination treatments is replacing single therapeutic protocols. Combination treatments involve non-artemisinins e.g., sulfadoxine+ pyrimethamine, sulfadoxine + pyrimethamine + amodiaquine, and artemisinins e.g., artesunate + amodiaquine, artesunate + mefloquine, artemether + lumefantrine, artesunate + sulfadoxine/pyrimethamine (Erhirhie et al., 2021). Curcumin-loaded liposomes combined with α/ β arteether showed therapeutic efficacy against malaria compared with liposome formulation alone; in addition, recrudescence was significantly prevented (Aditya et al., 2012). The Artemisinin-based combination therapies for uncomplicated Plasmodium falciparum infection in endemic regions have been recommended by the WHO e.g., Artemether-Lumefantrine (Art-L) (Coartem^®) (World Health Organization, 2015; Assefa et al., 2010). However, resistance among artemisinin and its partner drug continues to evolve, producing Plasmodium strains more capable of surviving treatment, which can subsequently spread across a wider geographical area. As a result, triple drug combinations mefloquine + dihydroartemisinin + piperaquine and amodiaquine + artemether + lumefantrine appeared to counterbalance the resistant mechanisms of the parasite; besides being well tolerated and safe. Adverse effects were mainly in the form of loss of appetite, nausea, and vomiting in some cases. Atovaquone + pyronaridine is also recommended in combination with different mechanisms of action (van der Pluijm et al., 2021).