Malaria antigen detection tests
Encyclopedia
Medical diagnosis
Medical diagnosis refers both to the process of attempting to determine or identify a possible disease or disorder , and to the opinion reached by this process...
of malaria
Malaria
Malaria is a mosquito-borne infectious disease of humans and other animals caused by eukaryotic protists of the genus Plasmodium. The disease results from the multiplication of Plasmodium parasites within red blood cells, causing symptoms that typically include fever and headache, in severe cases...
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
Antigen
An antigen is a foreign molecule that, when introduced into the body, triggers the production of an antibody by the immune system. The immune system will then kill or neutralize the antigen that is recognized as a foreign and potentially harmful invader. These invaders can be molecules such as...
suitable as target for Rapid Diagnostic Tests (RDTs) was a soluble glycolytic
Glycolysis
Glycolysis is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+...
enzyme
Enzyme
Enzymes are proteins that catalyze chemical reactions. In enzymatic reactions, the molecules at the beginning of the process, called substrates, are converted into different molecules, called products. Almost all chemical reactions in a biological cell need enzymes in order to occur at rates...
Glutamate dehydrogenase
Glutamate dehydrogenase
Glutamate dehydrogenase is an enzyme, present in most microbes and the mitochondria of eukaryotes, as are some of the other enzymes required for urea synthesis, that converts glutamate to α-Ketoglutarate, and vice versa. In animals, the produced ammonia is, however, usually bled off to the urea...
.
None of the rapid tests are currently as sensitive as a thick blood film
Blood film
A blood film or peripheral blood smear is a thin layer of blood smeared on a microscope slide and then stained in such a way to allow the various blood cells to be examined microscopically...
, nor as cheap. A major drawback in the use of all current dipstick
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....
methods is that the result is essentially qualitative. In many endemic areas of tropical Africa
Tropical Africa
Although tropical Africa is most familiar in the West as depicted by its rain forests, this region of Africa is far more diverse. While the tropics are thought of as regions with warm to hot moist climates caused by latitude and the tropical rain belt, the geology of areas, particularly mountain...
, 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 diseaseDisease
A disease is an abnormal condition affecting the body of an organism. It is often construed to be a medical condition associated with specific symptoms and signs. It may be caused by external factors, such as infectious disease, or it may be caused by internal dysfunctions, such as autoimmune...
if the patients have access to early diagnosis and prompt treatment
Therapy
This is a list of types of therapy .* Adventure therapy* Animal-assisted therapy* Aquatic therapy* Aromatherapy* Art and dementia* Art therapy* Authentic Movement* Behavioral therapy* Bibliotherapy* Buteyko Method* Chemotherapy...
. Antigen-based Rapid Diagnostic Tests (RDTs) have an important role at the periphery of health services capability because none of the rural clinic
Clinic
A clinic is a health care facility that is primarily devoted to the care of outpatients...
s has the ability to diagnose malaria on-site due to a lack of microscopes and trained technician
Technician
A technician is a worker in a field of technology who is proficient in the relevant skills and techniques, with a relatively practical understanding of the theoretical principles. Experienced technicians in a specific tool domain typically have intermediate understanding of theory and expert...
s to evaluate blood films. Furthermore, in regions where the disease is not endemic
Endemic (epidemiology)
In epidemiology, an infection is said to be endemic in a population when that infection is maintained in the population without the need for external inputs. For example, chickenpox is endemic in the UK, but malaria is not...
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
Tropics
The tropics is a region of the Earth surrounding the Equator. It is limited in latitude by the Tropic of Cancer in the northern hemisphere at approximately N and the Tropic of Capricorn in the southern hemisphere at S; these latitudes correspond to the axial tilt of the Earth...
and many of them return with a malaria infection. The RDT tests are still regarded as complements to conventional microscopy
Microscopy
Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...
but with some improvements it may well replace the microscope
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...
. The tests are simple and the procedure can be performed on the spot in field conditions. These tests use finger-stick or venous blood
Blood
Blood is a specialized bodily fluid in animals that delivers necessary substances such as nutrients and oxygen to the cells and transports metabolic waste products away from those same cells....
, the completed test takes a total of 15–20 minutes, and a laboratory 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
An accurate diagnosis is becoming more and more important, in view of the increasing resistance of Plasmodium falciparumPlasmodium falciparum
Plasmodium falciparum is a protozoan parasite, one of the species of Plasmodium that cause malaria in humans. It is transmitted by the female Anopheles mosquito. Malaria caused by this species is the most dangerous form of malaria, with the highest rates of complications and mortality...
and the high price of alternatives to chloroquine
Chloroquine
Chloroquine is a 4-aminoquinoline drug used in the treatment or prevention of malaria.-History:Chloroquine , N'--N,N-diethyl-pentane-1,4-diamine, was discovered in 1934 by Hans Andersag and co-workers at the Bayer laboratories who named it "Resochin". It was ignored for a decade because it was...
. The enzyme pGluDH does not occur in the host red blood cell
Red blood cell
Red blood cells are the most common type of blood cell and the vertebrate organism's principal means of delivering oxygen to the body tissues via the blood flow through the circulatory system...
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
China
Chinese civilization may refer to:* China for more general discussion of the country.* Chinese culture* Greater China, the transnational community of ethnic Chinese.* History of China* Sinosphere, the area historically affected by Chinese culture...
and is now undergoing clinical trial
Clinical trial
Clinical trials are a set of procedures in medical research and drug development that are conducted to allow safety and efficacy data to be collected for health interventions...
s.
GluDHs are ubiquitous enzymes that occupy an important branch-point between carbon and nitrogen metabolism. Both nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide
Nicotinamide adenine dinucleotide, abbreviated NAD, is a coenzyme found in all living cells. The compound is a dinucleotide, since it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine base and the other nicotinamide.In metabolism, NAD is involved...
(NAD) EC
EC number
The Enzyme Commission number is a numerical classification scheme for enzymes, based on the chemical reactions they catalyze....
1.4.1.2] and nicotinamide adenine dinucleotide phosphate
Nicotinamide adenine dinucleotide phosphate
Nicotinamide adenine dinucleotide phosphate, abbreviated NADP or TPN in older notation , is a coenzyme used in anabolic reactions, such as lipid and nucleic acid synthesis, which require NADPH as a reducing agent....
(NADP) dependent GluDH [EC 1.4.1.4] enzymes 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
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...
is a principal amino donor to other amino acids 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
Crystal structure
In mineralogy and crystallography, crystal structure is a unique arrangement of atoms or molecules in a crystalline liquid or solid. A crystal structure is composed of a pattern, a set of atoms arranged in a particular way, and a lattice exhibiting long-range order and symmetry...
is published. The GluDH activity in P.vivax
Plasmodium vivax
Plasmodium vivax is a protozoal parasite and a human pathogen. The most frequent and widely distributed cause of recurring malaria, P. vivax is one of the four species of malarial parasite that commonly infect humans. It is less virulent than Plasmodium falciparum, which is the deadliest of the...
, P.ovale
Plasmodium ovale
Plasmodium ovale is a species of parasitic protozoa that causes tertian malaria in humans. It is closely related to Plasmodium falciparum and Plasmodium vivax, which are responsible for most malaria. It is rare compared to these two parasites, and substantially less dangerous than P...
and P. malariae
Plasmodium malariae
Plasmodium malariae is a parasitic protozoa that causes malaria in humans. It is closely related to Plasmodium falciparum and Plasmodium vivax which are responsible for most malarial infection. While found worldwide, it is a so-called "benign malaria" and is not nearly as dangerous as that...
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
Isozyme
Isozymes are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes usually display different kinetic parameters Isozymes (also known as isoenzymes) are enzymes that differ in amino acid sequence but catalyze the same chemical reaction. These enzymes...
s, which allows strain differentiations (given the right monoclonal antibody
Monoclonal antibodies
Monoclonal antibodies are monospecific antibodies that are the same because they are made by identical immune cells that are all clones of a unique parent cell....
). 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 histidineHistidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
- and alanine
Alanine
Alanine is an α-amino acid with the chemical formula CH3CHCOOH. The L-isomer is one of the 20 amino acids encoded by the genetic code. Its codons are GCU, GCC, GCA, and GCG. It is classified as a nonpolar amino acid...
-rich, water-soluble protein, which is localized in several cell compartments including the parasite cytoplasm. The antigen is expressed only by P. falciparum trophozoites
Apicomplexan cellular morphology
Apicomplexa life cycle stages have evolved to allow intracellular parasites to survive the wide variety of environments they are exposed to during their complex life cycles. Each stage is typified by a cellular variety with a distinct morphology and biochemistry.Not all apicomplexa develop all the...
. HRP II from P. falciparum has been implicated in the biocrystallization
Biocrystallization
Biocrystallization is the formation of crystals from organic macromolecules by living organisms. This may be a stress response, a normal part of metabolism such as processes that dispose of waste compounds, or a pathology. Template mediated crystallization is qualitatively different from in vitro...
of hemozoin
Hemozoin
Hemozoin is a disposal product formed from the digestion of blood by some blood-feeding parasites. These hematophagous organisms such as Malaria parasites , Rhodnius and Schistosoma digest hemoglobin and release high quantities of free heme, which is the non-protein component of hemoglobin...
, 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 cell
Red blood cells are the most common type of blood cell and the vertebrate organism's principal means of delivering oxygen to the body tissues via the blood flow through the circulatory system...
, serum
Blood serum
In blood, the serum is the component that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed...
, plasma
Blood plasma
Blood plasma is the straw-colored liquid component of blood in which the blood cells in whole blood are normally suspended. It makes up about 55% of the total blood volume. It is the intravascular fluid part of extracellular fluid...
, cerebrospinal fluid
Cerebrospinal fluid
Cerebrospinal fluid , Liquor cerebrospinalis, is a clear, colorless, bodily fluid, that occupies the subarachnoid space and the ventricular system around and inside the brain and spinal cord...
and even urine
Urine
Urine is a typically sterile liquid by-product of the body that is secreted by the kidneys through a process called urination and excreted through the urethra. Cellular metabolism generates numerous by-products, many rich in nitrogen, that require elimination from the bloodstream...
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
Rheumatoid arthritis is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks synovial joints. The process produces an inflammatory response of the synovium secondary to hyperplasia of synovial cells, excess synovial fluid, and the development...
. Since HPR-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 dehydrogenaseLactate dehydrogenase
Lactate dehydrogenase is an enzyme present in a wide variety of organisms, including plants and animals.Lactate dehydrogenases exist in four distinct enzyme classes. Two of them are cytochrome c-dependent enzymes, each acting on either D-lactate or L-lactate...
(pLDH) is a 33 kDa oxidoreductase
Oxidoreductase
In biochemistry, an oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule to another...
[EC 1.1.1.27]. It is the last enzyme of the glycolytic pathway, essential for ATP
Adenosine triphosphate
Adenosine-5'-triphosphate is a multifunctional nucleoside triphosphate used in cells as a coenzyme. It is often called the "molecular unit of currency" of intracellular energy transfer. ATP transports chemical energy within cells for metabolism...
generation and one o the most abundant enzymes expressed by P.falciparum. PLDH does not persist in the blood but clears about the same time as the parasites following successful treatment. The lack of antigen persistence after treatment makes the pLDH test useful in predicting treatment failure. In this respect, pLDH is similar to pGluDH. LDH from P. vivax, P.malariae, and P.ovale exhibit 90-92% identity to pLDH from P.falciparum.
pAldo
Fructose-bisphosphate aldolaseFructose-bisphosphate aldolase
In molecular biology, fructose-bisphosphate aldolase , is a glycolytic enzyme that catalyses the reversible aldol cleavage or condensation of fructose-1,6-bisphosphate into dihydroxyacetone-phosphate and glyceraldehyde 3-phosphate...
[EC 4.1.2.13] catalyzes a key reaction in glycolysis
Glycolysis
Glycolysis is the metabolic pathway that converts glucose C6H12O6, into pyruvate, CH3COCOO− + H+...
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
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
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.