Malaria culture

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P. falciparum cultures Synchronsed P falciparum cultures.jpg
P. falciparum cultures

Malaria culture is a method for growing malaria parasites outside the body, i.e., in an ex vivo environment. Although attempts for propagation of the parasites outside of humans or animal models reach as far back as 1912, [2] the success of the initial attempts was limited to one or just a few cycles. The first successful continuous culture was established in 1976. [3] Initial hopes that the ex vivo culture would lead quickly to the discovery of a vaccine were premature. However, the development of new drugs was greatly facilitated. [4]

Contents

Method

Candlejar Trager & Jensen.jpg
Candlejar

Infected human red blood cells are incubated in a culture dish or flask at 37 °C together with a nutrient medium and plasma, serum or serum substitutes. [5] A special feature of the incubation is the special gas mixture filled with Nitrogen (90-92 %) CO2 (5 %) and Oxygen (3-5 %) allowing the parasites to grow at 37 °C in a cell incubator. [6] An alternative to gassing the cultures with the exact gas mixture, is the use of a candlejar. The candlejar is an airtight container in which the cultures and a lit candle are placed. The burning candle consumes some of the oxygen and produces carbon dioxide (CO2), which acts as a fire extinguisher. Carbon dioxide content in fresh air varies between 0.036 % and 0.039 %. Once the CO2 concentration reaches approximately 5 %, the candle stops burning. The number of parasites increased by a factor 5 approximately every 48 hours (one cycle). The parasitemia can be determined via blood film, to keep it within the wanted limits, the culture can be thinned out with healthy red blood cells. [7]

Concentration of P. falciparum-infected erythrocytes by discontinuous density gradient centrifugation in Percoll. Gradient centrifugation of infected Red Blood Cells.jpg
Concentration of P. falciparum-infected erythrocytes by discontinuous density gradient centrifugation in Percoll.

The original method for the successful ex vivo propagation of P. falciparum described culture of the parasite under static conditions (Trager-Jensen method). [3] James B. Jensen joined Trager’s laboratory as a post-doctoral fellow in 1976. He decided to employ a candlejar instead of the CO2 incubator. In the summer of 1976 Milton Friedman, a graduate student in the Trager lab who was working in the MRC laboratories in The Gambia, arranged for a sample of human blood infected with P. falciparum to be sent to New York City. This was diluted with RPMI 1640 (which turned out to be the best of the commercial media) in Petri dishes, placed in a candlejar and incubated. The line grew very well and became FCR-3/Gambia, one of the most widely used strains. Later, other lines would be established using similar methods and the impact of continuous cultivation of P. falciparum was phenomenal especially for the testing of putative antimalarials and for deciphering its genes. A number of subsequent reports (from as far back as the early 1980s), showed that cell suspension (using a shaking-incubator) significantly increased culture growth. Continuous agitation has also been shown to improve other parameters of culture growth relevant to researchers, such as the prolongation of culture synchrony after synchronization procedures, and a reduction of the rate of multiple infections. [9] Despite this, the practice of culturing the parasite under static conditions remains widespread. The greatest value of the candlejar method is that it can be used in laboratories almost anywhere in the world where there is an incubator, a candle and a desiccator. [10] Around 60% parasitized cells can be obtained using optimized culturing conditions. [1] Recent studies of P. falciparum isolated directly from infected patients indicate that alternative parasite biological states occur in the natural host that are not observed with ex vivo cultivated parasites. [11]

Concentration of infected cells

Blood stages of P.falciparum Blood stages of P. falciparum.jpg
Blood stages of P.falciparum
Magnetic collection of P.falciparum infected blood Magnetic collection.jpg
Magnetic collection of P.falciparum infected blood

To achieve synchronization and/or concentration of the parasites in culture several methods have been developed. A discontinuous Percoll gradient procedure can be used to isolate infected red blood cells because red cells containing plasmodia are less dense than normal ones. Young trophozoites coincided with erythrocytes in a broad band corresponding to densities from 1.075 to 1.100 g/ml, whereas schizonts were concentrated at a density approximating 1.062 g/ml. [14] There are studies, however, that suggest that some strains of P.falciparum are affected in their capacity of invasion after being exposed to this chemical. The difference between diamagnetic low-spin oxyhemoglobin in uninfected red blood cells and paramagnetic hemozoin in infected red blood cells can also be used for isolation. Magnetic columns have shown to be less harmful for the parasite and are simple and adjustable to the needs of the researcher. [15] [16] The column is mounted in a potent magnet holder and the culture flowed through it. The column traps the erythrocytes infected with the latest stages of the parasites, which can then be eluted when the column is removed from the magnet. It is a simple method that does not need expensive equipment and it does not seem to affect the parasites as to their invasion capabilities afterwards. [12]

Related Research Articles

<span class="mw-page-title-main">Malaria</span> Mosquito-borne infectious disease

Malaria is a mosquito-borne infectious disease that affects humans and other vertebrates. Human malaria causes symptoms that typically include fever, fatigue, vomiting, and headaches. In severe cases, it can cause jaundice, seizures, coma, or death. Symptoms usually begin 10 to 15 days after being bitten by an infected Anopheles mosquito. If not properly treated, people may have recurrences of the disease months later. In those who have recently survived an infection, reinfection usually causes milder symptoms. This partial resistance disappears over months to years if the person has no continuing exposure to malaria.

<i>Plasmodium</i> Genus of parasitic protists that can cause malaria

Plasmodium is a genus of unicellular eukaryotes that are obligate parasites of vertebrates and insects. The life cycles of Plasmodium species involve development in a blood-feeding insect host which then injects parasites into a vertebrate host during a blood meal. Parasites grow within a vertebrate body tissue before entering the bloodstream to infect red blood cells. The ensuing destruction of host red blood cells can result in malaria. During this infection, some parasites are picked up by a blood-feeding insect, continuing the life cycle.

<i>Plasmodium falciparum</i> Protozoan species of malaria parasite

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 disease's most dangerous form, falciparum malaria. It is responsible for around 50% of all malaria cases. P. falciparum is therefore regarded as the deadliest parasite in humans. It is also associated with the development of blood cancer and is classified as a Group 2A (probable) carcinogen.

<span class="mw-page-title-main">Gametocyte</span> Eukaryotic germ stem cell

A gametocyte is a eukaryotic germ cell that divides by mitosis into other gametocytes or by meiosis into gametids during gametogenesis. Male gametocytes are called spermatocytes, and female gametocytes are called oocytes.

<i>Plasmodium vivax</i> Species of single-celled organism

Plasmodium vivax is a protozoal parasite and a human pathogen. This parasite is the most frequent and widely distributed cause of recurring malaria. Although it is less virulent than Plasmodium falciparum, the deadliest of the five human malaria parasites, P. vivax malaria infections can lead to severe disease and death, often due to splenomegaly. P. vivax is carried by the female Anopheles mosquito; the males do not bite.

<i>Plasmodium ovale</i> Species of single-celled organism

Plasmodium ovale is a species of parasitic protozoon that causes tertian malaria in humans. It is one of several species of Plasmodium parasites that infect humans, including Plasmodium falciparum and Plasmodium vivax which are responsible for most cases of malaria in the world. P. ovale is rare compared to these two parasites, and substantially less dangerous than P. falciparum.

<i>Plasmodium malariae</i> Species of single-celled organism

Plasmodium malariae is a parasitic protozoan that causes malaria in humans. It is one of several species of Plasmodium parasites that infect other organisms as pathogens, also including Plasmodium falciparum and Plasmodium vivax, responsible for most malarial infection. Found worldwide, it causes a so-called "benign malaria", not nearly as dangerous as that produced by P. falciparum or P. vivax. The signs include fevers that recur at approximately three-day intervals – a quartan fever or quartan malaria – longer than the two-day (tertian) intervals of the other malarial parasite.

<i>Plasmodium knowlesi</i> Species of single-celled organism

Plasmodium knowlesi is a parasite that causes malaria in humans and other primates. It is found throughout Southeast Asia, and is the most common cause of human malaria in Malaysia. Like other Plasmodium species, P. knowlesi has a life cycle that requires infection of both a mosquito and a warm-blooded host. While the natural warm-blooded hosts of P. knowlesi are likely various Old World monkeys, humans can be infected by P. knowlesi if they are fed upon by infected mosquitoes. P. knowlesi is a eukaryote in the phylum Apicomplexa, genus Plasmodium, and subgenus Plasmodium. It is most closely related to the human parasite Plasmodium vivax as well as other Plasmodium species that infect non-human primates.

<i>Plasmodium berghei</i> Single celled parasite, rodent malaria

Plasmodium berghei is a single-celled parasite causing rodent malaria. It is in the Plasmodium subgenus Vinckeia.

<span class="mw-page-title-main">Malaria antigen detection tests</span>

Malaria antigen detection tests are a group of commercially available rapid diagnostic tests of the rapid antigen test type that allow quick diagnosis of malaria 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. The first malaria antigen suitable as target for such a test was a soluble glycolytic enzyme Glutamate dehydrogenase. None of the rapid tests are currently as sensitive as a thick blood film, nor as cheap. A major drawback in the use of all current dipstick methods is that the result is essentially qualitative. In many endemic areas of tropical Africa, however, the quantitative assessment of parasitaemia is important, as a large percentage of the population will test positive in any qualitative assay.

Malaria vaccines are vaccines that prevent malaria, a mosquito-borne infectious disease which annually affects an estimated 247 million people worldwide and causes 619,000 deaths. The first approved vaccine for malaria is RTS,S, known by the brand name Mosquirix. As of April 2023, the vaccine has been given to 1.5 million children living in areas with moderate-to-high malaria transmission. It requires at least three doses in infants by age 2, and a fourth dose extends the protection for another 1–2 years. The vaccine reduces hospital admissions from severe malaria by around 30%.

<span class="mw-page-title-main">History of malaria</span> History of malaria infections

The history of malaria extends from its prehistoric origin as a zoonotic disease in the primates of Africa through to the 21st century. A widespread and potentially lethal human infectious disease, at its peak malaria infested every continent except Antarctica. Its prevention and treatment have been targeted in science and medicine for hundreds of years. Since the discovery of the Plasmodium parasites which cause it, research attention has focused on their biology as well as that of the mosquitoes which transmit the parasites.

<span class="mw-page-title-main">Hemozoin</span>

Haemozoin 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 haemoglobin and release high quantities of free heme, which is the non-protein component of haemoglobin. Heme is a prosthetic group consisting of an iron atom contained in the center of a heterocyclic porphyrin ring. Free heme is toxic to cells, so the parasites convert it into an insoluble crystalline form called hemozoin. In malaria parasites, hemozoin is often called malaria pigment.

Human genetic resistance to malaria refers to inherited changes in the DNA of humans which increase resistance to malaria and result in increased survival of individuals with those genetic changes. The existence of these genotypes is likely due to evolutionary pressure exerted by parasites of the genus Plasmodium which cause malaria. Since malaria infects red blood cells, these genetic changes are most common alterations to molecules essential for red blood cell function, such as hemoglobin or other cellular proteins or enzymes of red blood cells. These alterations generally protect red blood cells from invasion by Plasmodium parasites or replication of parasites within the red blood cell.

Nycteria is a genus of protozoan parasites that belong to the phylum Apicomplexa. It is composed of vector-borne haemosporidian parasites that infect a wide range of mammals such as primates, rodents and bats. Its vertebrate hosts are bats. First described by Garnham and Heisch in 1953, Nycteria is mostly found in bat species where it feeds off the blood of their hosts and causes disease. Within the host, Nycteria develops into peculiar lobulated schizonts in parenchyma cells of the liver, similarly to the stages of Plasmodium falciparum in the liver. The vector of Nycteria has been hard to acquire and identify. Because of this, the life cycle of Nycteria still remains unknown and understudied. It has been suggested that this vector could be an arthropod other than a mosquito or the vector of most haemosporidian parasites.

The mainstay of malaria diagnosis has been the microscopic examination of blood, utilizing blood films. Although blood is the sample most frequently used to make a diagnosis, both saliva and urine have been investigated as alternative, less invasive specimens. More recently, modern techniques utilizing antigen tests or polymerase chain reaction have been discovered, though these are not widely implemented in malaria endemic regions. Areas that cannot afford laboratory diagnostic tests often use only a history of subjective fever as the indication to treat for malaria.

Plasmodium coatneyi is a parasitic species that is an agent of malaria in nonhuman primates. P. coatneyi occurs in Southeast Asia. The natural host of this species is the rhesus macaque and crab-eating macaque, but there has been no evidence that zoonosis of P. coatneyi can occur through its vector, the female Anopheles mosquito.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a family of proteins present on the membrane surface of red blood cells that are infected by the malarial parasite Plasmodium falciparum. PfEMP1 is synthesized during the parasite's blood stage inside the RBC, during which the clinical symptoms of falciparum malaria are manifested. Acting as both an antigen and adhesion protein, it is thought to play a key role in the high level of virulence associated with P. falciparum. It was discovered in 1984 when it was reported that infected RBCs had unusually large-sized cell membrane proteins, and these proteins had antibody-binding (antigenic) properties. An elusive protein, its chemical structure and molecular properties were revealed only after a decade, in 1995. It is now established that there is not one but a large family of PfEMP1 proteins, genetically regulated (encoded) by a group of about 60 genes called var. Each P. falciparum is able to switch on and off specific var genes to produce a functionally different protein, thereby evading the host's immune system. RBCs carrying PfEMP1 on their surface stick to endothelial cells, which facilitates further binding with uninfected RBCs, ultimately helping the parasite to both spread to other RBCs as well as bringing about the fatal symptoms of P. falciparum malaria.

<i>Plasmodium</i> helical interspersed subtelomeric protein

The Plasmodium helical interspersed subtelomeric proteins (PHIST) or ring-infected erythrocyte surface antigens (RESA) are a family of protein domains found in the malaria-causing Plasmodium species. It was initially identified as a short four-helical conserved region in the single-domain export proteins, but the identification of this part associated with a DnaJ domain in P. falciparum RESA has led to its reclassification as the RESA N-terminal domain. This domain has been classified into three subfamilies, PHISTa, PHISTb, and PHISTc.

<span class="mw-page-title-main">David A. Fidock</span>

David A. Fidock, is the CS Hamish Young Professor of Microbiology and Immunology and Professor of Medical Sciences at Columbia University Irving Medical Center in Manhattan.

References

  1. 1 2 3 Radfar A, Méndez D, Moneriz C, Linares M, Marín-García P, Puyet A, Diez A, Bautista JM (2009). "Synchronous culture of Plasmodium falciparum at high parasitemia levels". Nat. Protoc. 4 (11): 1899–915. doi:10.1038/nprot.2009.198. PMID   20010926.
  2. ↑ Bass CC, Johns FM. (1912). "The Cultivation of Malarial Plasmodia (Plasmodium vivax and Plasmodium Falciparum) in vitro". J. Exp. Med. 16 (4): 567–579. doi:10.1084/jem.16.4.567. PMC   2124976 . PMID   19867597.
  3. 1 2 Trager W, Jensen JB (1976). "Human malaria parasites in continuous culture". Science. 193 (4254): 673–675. Bibcode:1976Sci...193..673T. doi:10.1126/science.781840. PMID   781840.
  4. ↑ Trager W, Jensen JB (1997). "Continuous culture of Plasmodium falciparum: its impact on malaria research". Int. J. Parasitol. 27 (9): 989–1006. doi:10.1016/S0020-7519(97)00080-5. PMID   9363481.
  5. ↑ Basco LK (2003). "Molecular epidemiology of malaria in Cameroon. XV. Experimental studies on serum substitutes and supplements and alternative culture media for in vitro drug sensitivity assays using fresh isolates of Plasmodium falciparum". Am. J. Trop. Med. Hyg. 69 (2): 168–173. doi: 10.4269/ajtmh.2003.69.168 . PMID   14506772.
  6. ↑ Trigg PI (1985). "Recent advances in malaria parasite cultivation and their application to studies on host-parasite relationships: a review" (PDF). Bull. World Health Organ. 63 (2): 387–398. PMC   2536397 . PMID   3893779. Archived from the original (PDF) on November 28, 2003.
  7. ↑ Schuster FL (2002). "Cultivation of plasmodium spp". Clin. Microbiol. Rev. 15 (3): 355–364. doi:10.1128/CMR.15.3.355-364.2002. PMC   118084 . PMID   12097244.
  8. ↑ Rüssmann L, Jung A, Heidrich HG (1982). "The use of percoll gradients, elutriator rotor elution, and mithramycin staining for the isolation and identification of intraerythrocytic stages of plasmodium berghei". Z. Parasitenkd. 66 (3): 273–280. doi:10.1007/BF00925344. PMID   6177116.
  9. ↑ Allen RJ, Kirk K (2010). "Plasmodium falciparum culture: The benefits of shaking". Mol. Biochem. Parasitol. 169 (1): 63–5. doi:10.1016/j.molbiopara.2009.09.005. PMID   19766147.
  10. ↑ Sherman, I. W. (2010). Magic Bullets to Conquer Malaria. From Quinine to Qinghaosu. ASM Press. ISBN   978-1-55581-543-1.
  11. ↑ LeRoux M, Lakshmanan V, Daily JP (2009). "Plasmodium falciparum biology: analysis of in vitro versus in vivo growth conditions". Trends Parasitol. 25 (10): 474–481. doi:10.1016/j.pt.2009.07.005. PMID   19747879.
  12. 1 2 Spadafora C, Gerena L, Kopydlowski KM (2011). "Comparison of the in vitro invasive capabilities of Plasmodium falciparum schizonts isolated by Percoll gradient or using magnetic based separation". Malar. J. 10 (1): 96. doi: 10.1186/1475-2875-10-96 . PMC   3100259 . PMID   21501476.
  13. ↑ Coronado LM, Tayler NM, Correa R, Giovani RM, Spadafora C (2013). "Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means". J. Vis. Exp. 73 (73): e50342. doi:10.3791/50342. PMC   3622091 . PMID   23486405.
  14. ↑ Rivadeneira EM, Wasserman M, Espinal CT (1983). "Separation and concentration of schizonts of Plasmodium falciparum by Percoll gradients". J. Protozool. 30 (2): 367–370. doi:10.1111/j.1550-7408.1983.tb02932.x. PMID   6313915.[ dead link ]
  15. ↑ Kim CC, Wilson EB, Derisi JL (2010). "Improved methods for magnetic purification of malaria parasites and haemozoin" (PDF). Malar. J. 9 (1): 17. doi: 10.1186/1475-2875-9-17 . PMC   2817699 . PMID   20074366.
  16. ↑ Bhakdi SC, Ottinger A, Somsri S, Sratogno P, Pannadaporn P, Chimma P, Malasit P, Pattanapanyasat K, Neumann HP (2010). "Optimized high gradient magnetic separation for isolation of Plasmodium-infected red blood cells" (PDF). Malar. J. 9 (1): 38. doi: 10.1186/1475-2875-9-38 . PMC   2831011 . PMID   20122252.

Further reading

• Doolan, D. L. (Editor) (2002) Malaria Methods and Protocols (Methods in Molecular Medicine) , Totowa, NJ: Humana Press, ISBN   0-89603-823-8 / ISBN   978-0-89603-823-3

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