Malaria antigen detection tests are a group of commercially available

rapid diagnostic test A rapid diagnostic test (RDT) is a medical diagnostic test that is quick and easy to perform. RDTs are suitable for preliminary or emergency medical screening and for use in medical facilities with limited resources. They also allow point-of-care ...

s of the

rapid antigen test A rapid antigen test (RAT), sometimes called a rapid antigen detection test (RADT), antigen rapid test (ART), or loosely just a rapid test, is a rapid diagnostic test suitable for point-of-care testing that directly detects the presence or absenc ...

type that allow quick

diagnosis Diagnosis is the identification of the nature and cause of a certain phenomenon. Diagnosis is used in many different disciplines, with variations in the use of logic, analytics, and experience, to determine " cause and effect". In systems engine ...

malaria Malaria is a Mosquito-borne disease, mosquito-borne infectious disease that affects humans and other animals. Malaria causes Signs and symptoms, symptoms that typically include fever, fatigue (medical), tiredness, vomiting, and headaches. In se ...

by people who are not otherwise skilled in traditional laboratory techniques for diagnosing malaria or in situations where such equipment is not available. There are currently over 20 such tests commercially available (WHO product testing 2008). The first malaria

antigen In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. ...

suitable as target for such a test was a soluble

glycolytic Glycolysis is the metabolic pathway that converts glucose () into pyruvate (). The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) ...

enzyme Enzymes () are proteins that act as biological catalysts by accelerating chemical reactions. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. ...

Glutamate dehydrogenase Glutamate dehydrogenase (GLDH, GDH) is an enzyme observed in both prokaryotes and eukaryotic mitochondria. The aforementioned reaction also yields ammonia, which in eukaryotes is canonically processed as a substrate in the urea cycle. Typical ...

. None of the rapid tests are currently as sensitive as a thick

blood film A blood smear, peripheral blood smear or blood film is a thin layer of blood smeared on a glass microscope slide and then stained in such a way as to allow the various blood cells to be examined microscopically. Blood smears are examined in th ...

, nor as cheap. A major drawback in the use of all current

dipstick A dipstick is one of several measurement devices. Some dipsticks are dipped into a liquid to perform a chemical test or to provide a measure of quantity of the liquid. Since the late 20th century, a flatness/levelness measuring device trademarke ...

methods is that the result is essentially qualitative. In many endemic areas of

tropical Africa Although tropical Africa is mostly familiar to the West for its rainforests, this biogeographic realm of Africa is far more diverse. While the tropics are thought of as regions with hot moist climates, which are caused by latitude and the trop ...

, however, the quantitative assessment of parasitaemia is important, as a large percentage of the population will test positive in any qualitative assay.

Antigen-based Malaria Rapid Diagnostic Tests

Malaria is a curable

disease A disease is a particular abnormal condition that negatively affects the structure or function of all or part of an organism, and that is not immediately due to any external injury. Diseases are often known to be medical conditions that ar ...

if the patients have access to early diagnosis and prompt treatment. Antigen-based

(RDTs)
have an important role at the periphery of health services capability because many rural

clinic A clinic (or outpatient clinic or ambulatory care clinic) is a health facility that is primarily focused on the care of outpatients. Clinics can be privately operated or publicly managed and funded. They typically cover the primary care need ...

s do not have the ability to diagnose malaria on-site due to a lack of microscopes and trained

technician A technician is a worker in a field of technology who is proficient in the relevant skill and technique, with a relatively practical understanding of the theoretical principles. Specialisation The term technician covers many different special ...

s to evaluate blood films. Furthermore, in regions where the disease is not

endemic Endemism is the state of a species being found in a single defined geographic location, such as an island, state, nation, country or other defined zone; organisms that are indigenous to a place are not endemic to it if they are also found else ...

, laboratory technologists have very limited experience in detecting and identifying malaria parasites. An ever increasing numbers of travelers from temperate areas each year visit tropical countries and many of them return with a malaria infection. The RDT tests are still regarded as complements to conventional

microscopy Microscopy is the technical field of using microscopes to view objects and areas of objects that cannot be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of mic ...

but with some improvements it may well replace the microscope. The tests are simple and the procedure can be performed on the spot in field conditions. These tests use finger-stick or venous blood, the completed test takes a total of 15–20 minutes, and a

laboratory A laboratory (; ; colloquially lab) is a facility that provides controlled conditions in which scientific or technological research, experiments, and measurement may be performed. Laboratory services are provided in a variety of settings: physicia ...

is not needed. The threshold of detection by these rapid diagnostic tests is in the range of 100 parasites/µl of blood compared to 5 by thick film microscopy.

pGluDH

Plasmodium glutamate dehydrogenase precipitation

An accurate diagnosis is becoming more and more important, in view of the increasing resistance of ''

Plasmodium falciparum ''Plasmodium falciparum'' is a unicellular protozoan parasite of humans, and the deadliest species of ''Plasmodium'' that causes malaria in humans. The parasite is transmitted through the bite of a female ''Anopheles'' mosquito and causes the di ...

'' and the high price of alternatives to

chloroquine Chloroquine is a medication primarily used to prevent and treat malaria in areas where malaria remains sensitive to its effects. Certain types of malaria, resistant strains, and complicated cases typically require different or additional medi ...

. The enzyme pGluDH does not occur in the host

red blood cell Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...

and was recommended as a marker enzyme for ''Plasmodium'' species by Picard-Maureau et al. in 1975. The malaria marker enzyme test is suitable for routine work and is now a standard test in most departments dealing with malaria. Presence of pGluDH is known to represent parasite viability and a rapid diagnostic test using pGluDH as antigen would have the ability to differentiate live from dead organisms. A complete RDT with pGluDH as antigen has been developed in China and is now undergoing

clinical trial Clinical trials are prospective biomedical or behavioral research studies on human subject research, human participants designed to answer specific questions about biomedical or behavioral interventions, including new treatments (such as novel v ...

s. GluDHs are ubiquitous enzymes that occupy an important branch-point between carbon and nitrogen metabolism. Both

nicotinamide adenine dinucleotide Nicotinamide adenine dinucleotide (NAD) is a coenzyme central to metabolism. Found in all living cells, NAD is called a dinucleotide because it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an ade ...

(NAD) C 1.4.1.2and

nicotinamide adenine dinucleotide phosphate Nicotinamide adenine dinucleotide phosphate, abbreviated NADP or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle and lipid and nucleic acid syntheses, which require NA ...

(NADP) dependent GluDH C 1.4.1.4enzymes are present in ''Plasmodia''; the NAD-dependent GluDH is relatively unstable and not useful for diagnostic purposes. Glutamate dehydrogenase provides an oxidizable carbon source used for the production of energy as well as a reduced electron carrier, NADH.

Glutamate Glutamic acid (symbol Glu or E; the ionic form is known as glutamate) is an α-amino acid that is used by almost all living beings in the biosynthesis of proteins. It is a non-essential nutrient for humans, meaning that the human body can syn ...

is a principal amino donor to other

amino acids Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...

in subsequent transamination reactions. The multiple roles of glutamate in nitrogen balance make it a gateway between free ammonia and the amino groups of most amino acids. Its crystal structure is published. The GluDH activity in '' P.vivax'', '' P.ovale'' and '' P. malariae'' has never been tested, but given the importance of GluDH as a branch point enzyme, every cell must have a high concentration of GluDH. It is well known that enzymes with a high molecular weight (like GluDH) have many

isozyme In biochemistry, isozymes (also known as isoenzymes or more generally as multiple forms of enzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. Isozymes usually have different kinetic parameters (e.g. dif ...

s, which allows strain differentiations (given the right monoclonal antibody). The host produces antibodies against the parasitic enzyme indicating a low sequence identity.

Histidine rich protein II

The histidine-rich protein II (HRP II) is a

histidine Histidine (symbol His or H) is an essential amino acid that is used in the biosynthesis of proteins. It contains an α-amino group (which is in the protonated –NH3+ form under biological conditions), a carboxylic acid group (which is in the d ...

- and

alanine Alanine (symbol Ala or A), or α-alanine, is an α-amino acid that is used in the biosynthesis of proteins. It contains an amine group and a carboxylic acid group, both attached to the central carbon atom which also carries a methyl group side ...

-rich, water-soluble protein, which is localized in several cell compartments including the parasite cytoplasm. The antigen is expressed only by ''P. falciparum''

trophozoites A trophozoite (G. ''trope'', nourishment + ''zoon'', animal) is the activated, feeding stage in the life cycle of certain protozoa such as malaria-causing ''Plasmodium falciparum'' and those of the ''Giardia'' group. (The complement of the trophozoi ...

. HRP II from ''P. falciparum'' has been implicated in the biocrystallization of

hemozoin Haemozoin is a disposal product formed from the digestion of blood by some blood-feeding parasites. These hematophagous organisms such as malaria parasites ('' Plasmodium spp.''), '' Rhodnius'' and '' Schistosoma'' digest haemoglobin and release ...

, an inert, crystalline form of ferriprotoporphyrin IX (Fe(3+)-PPIX) produced by the parasite. A substantial amount of the HRP II is secreted by the parasite into the host bloodstream and the antigen can be detected in

erythrocytes Red blood cells (RBCs), also referred to as red cells, red blood corpuscles (in humans or other animals not having nucleus in red blood cells), haematids, erythroid cells or erythrocytes (from Greek ''erythros'' for "red" and ''kytos'' for "holl ...

, serum, plasma,

cerebrospinal fluid Cerebrospinal fluid (CSF) is a clear, colorless body fluid found within the tissue that surrounds the brain and spinal cord of all vertebrates. CSF is produced by specialised ependymal cells in the choroid plexus of the ventricles of the b ...

and even

urine Urine is a liquid by-product of metabolism in humans and in many other animals. Urine flows from the kidneys through the ureters to the urinary bladder. Urination results in urine being excreted from the body through the urethra. Cellular m ...

as a secreted water-soluble protein. These antigens persist in the circulating blood after the parasitaemia has cleared or has been greatly reduced. It generally takes around two weeks after successful treatment for HRP2-based tests to turn negative, but may take as long as one month, which compromises their value in the detection of active infection. False positive dipstick results were reported in patients with rheumatoid-factor-positive

rheumatoid arthritis Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects joints. It typically results in warm, swollen, and painful joints. Pain and stiffness often worsen following rest. Most commonly, the wrist and hands are involv ...

. Since HRP-2 is expressed only by ''P. falciparum'', these tests will give negative results with samples containing only ''P. vivax'', ''P. ovale'', or ''P. malariae''; many cases of non-falciparum malaria may therefore be misdiagnosed as malaria negative (some ''P.falciparum'' strains also don't have HRP II). The variability in the results of pHRP2-based RDTs is related to the variability in the target antigen.

pLDH

''P. falciparum''

lactate dehydrogenase Lactate dehydrogenase (LDH or LD) is an enzyme found in nearly all living cells. LDH catalyzes the conversion of lactate to pyruvate and back, as it converts NAD+ to NADH and back. A dehydrogenase is an enzyme that transfers a hydride from one ...

(PfLDH) is a 33 kDa

oxidoreductase In biochemistry, an oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule, the reductant, also called the electron donor, to another, the oxidant, also called the electron acceptor. This group of enzymes usually ut ...

C 1.1.1.27 It is the last enzyme of the glycolytic pathway, essential for ATP generation and one of the most abundant enzymes expressed by ''P. falciparum''. ''Plasmodium'' LDH (pLDH) from ''P. vivax'', ''P. malariae'', and ''P. ovale'') exhibit 90-92% identity to PfLDH from ''P. falciparum''. pLDH levels have been seen to reduce in the blood sooner after treatment than HRP2. In this respect, pLDH is similar to pGluDH. Nevertheless, the kinetic properties and sensitivities to inhibitors targeted to the cofactor binding site differ significantly and are identifiable by measuring dissociation constants for inhibitors which, differ by up to 21-fold.

pAldo

Fructose-bisphosphate aldolase Fructose-bisphosphate aldolase (), often just aldolase, is an enzyme catalyzing a reversible reaction that splits the aldol, fructose 1,6-bisphosphate, into the triose phosphates dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate ( ...

C 4.1.2.13catalyzes a key reaction in

glycolysis Glycolysis is the metabolic pathway that converts glucose () into pyruvate (). The free energy released in this process is used to form the high-energy molecules adenosine triphosphate (ATP) and reduced nicotinamide adenine dinucleotide (NADH) ...

and energy production and is produced by all four species. The ''P.falciparum'' aldolase is a 41 kDa protein and has 61-68% sequence similarity to known

eukaryotic Eukaryotes () are organisms whose cells have a nucleus. All animals, plants, fungi, and many unicellular organisms, are Eukaryotes. They belong to the group of organisms Eukaryota or Eukarya, which is one of the three domains of life. Bact ...

aldolases. Its crystal structure has been published. The presence of antibodies against p41 in the sera of human adults partially immune to malaria suggest that p41 is implicated in protective immune response against the parasite.

References

External links

Malaria Antibodies

WHO product testing 2008

WHO Rapid Diagnostic Tests (RDTs)
{{malaria Malaria Blood tests