Malaria is caused by protozoan parasites called Plasmodia, belonging to the parasitic phylum Apicomplexa. More than 200 species of the genus Plasmodium (=plasma + eidos, form) have been identified that are parasitic to reptiles, birds, and mammals.[1] Four Plasmodium species have been well known to cause human malaria, namely, P. falciparum, P. vivax, P. ovale, and P. malariae. A fifth one, P. knowlesi, has been recently documented to cause human infections in many countries of Southeast Asia.[2] Very rare cases of malaria have been reported due to other species such as Plasmodium brasilianum, Plasmodium cynomolgi, Plasmodium cynomolgi bastianellii, Plasmodium inui, Plasmodium rhodiani, Plasmodium schwetzi, Plasmodium semiovale, Plasmodium simium and Plasmodium eylesi. All malaria parasites infecting humans probably jumped from the great apes (in case of P. knowlesi, macaques) to man.[See]

Phylum: Protozoa Subphylum: Apicomplexa

Class: Sporozoa Subclass: Coccidia

Order: Coccidiida Suborder: Haemosporina

Family: Plasmodiidae

Genus: Plasmodia

Subgenera: Plasmodium, Laverania

Species: (affecting man)

Quartan group: P. (Plasmodium) malariae, P. (P.) brasilianum

Benign tertian group: P. (P.) vivax, P. (P.) cynomolgi, P. (P.) cynomolgi bastianellii

Malignant tertian group: P. (Laverania) falciparum

Ovale group: P. (P.) ovale, P. (P.) simium

Knowlesi group: P. (P.) knowlesi

Phylogenetic Trees of Plasmodia

Source: Krief S et al. On the Diversity of Malaria Parasites in African Apes and the Origin of P. falciparum from Bonobos. PLoS Pathog 2010;6(2): e1000765.[Full Text]

Subtypes of P. vivax: Plasmodium vivax is divided into two subtypes, a dominant form, VK210 and a variant form, VK247. This division is dependent on the amino acid composition of the circumsporozoite (CS) protein. A strain of P. vivax containing a variant repeat in its CS protein was first isolated in Thailand.[3,4]. The CS repeat of this variant strain (Thai VK247) differs at 6/9 amino acids within the repeat sequence found in all previously described P. vivax CS protein. Following this discovery, several studies have been conducted to evaluate the global distribution of variant VK247; it was detected in indigenous populations of China [5], Brazil [6], Mexico [7,8], Peru [8,9], and Papua New Guinea [8]. It is known that the drug susceptibility of the VK247 subtype of P. vivax is slightly different than VK210 [10], as well as that Anopheles albimanus and Anopheles pseudopunctipennis differ in their susceptibilities to P. vivax circumsporozoite phenotypes. Anopheles albimanus is more susceptible to the VK210 subtype, whereas An. pseudopunctipennis is more susceptible to the VK247 subtype.[11]

Two species of P. ovale: P. ovale has been found to exist in two forms, classic and variant, with the latter accounting for the higher parasite density among humans. A new study has now proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasite, Plasmodium ovale curtisi (classic type) and Plasmodium ovale wallikeri (variant type), named so in honor of malaria researchers Christopher F. Curtis (1939-2008) and David Walliker (1940-2007). These two nonrecombining, genetically distinct species coexist, being sympatric in Africa and Asia. Splitting of the 2 lineages is estimated to have occurred between 1.0 and 3.5 million years ago in hominid hosts.[12-16] [More at The Lancet]

Molecular characteristics of malarial parasites have also been studied in India.[17]

Distribution of Plasmodia: Nearly 85% of cases in Africa are caused by P. falciparum, the remaining cases being caused by the other three strains. P. vivax is now the most geographically widespread of the human malarias, occurring in much of Asia, Central and South America, the Middle East, where 70–90% of the malaria burden is of this species and the rest due to P. falciparum.[1,18] P. malariae causes sporadic infections in Africa, parts of India, western Pacific and South America, whereas P. ovale is restricted to tropical Africa, New Guinea, and the Philippines.[18] P. knowlesi has been reported from South East Asian countries such as Malaysia, Thailand, Viet Nam, Myanmar and Phillippines.[19-23]

Analyses of the mtDNA data have revealed that P. knowlesi is derived from an ancestral parasite population that existed prior to human settlement in Southeast Asia, and underwent significant population expansion approximately 30,000–40,000 years ago. The results indicate that human infections with P. knowlesi are not newly emergent in Southeast Asia and that knowlesi malaria is primarily a zoonosis with wild macaques as the reservoir hosts. Ongoing ecological changes resulting from deforestation, with an associated increase in the human population, could enable this pathogenic species of Plasmodium to switch to humans as the preferred host.[24]

Comparison of Malaria Parasites [21,25-32]
	P. falciparum	P. vivax	P. ovale	P. malariae	P. knowlesi
Global Distribution	80-90% of cases in Africa, 40-50% of cases in western pacific and SE Asia, 4-30% in S Asia, S America and rest of tropics	70-90% of cases in most of Asia and S America, 50-60% of cases in SE Asia and western pacific, 1-10% in Africa	8% of cases in parts of Africa, stray cases in Asia	2-3% in Africa, sporadic in Asia and S America	Reported from SE Asia; 70% of cases in some of those areas
Occurrence in India	30–90% of cases in Orissa, the NE states, Chattisgarh, Jharkhand, Madhya Pradesh, Bihar, and Andamans; <10% of cases in other areas	Nearly 50% of total malaria burden; predominant species in most parts other than P. falciparum dominant areas.	Stray cases reported from Delhi, Gujarat Kolkata, Orissa, and Assam	3-16% reported from some tribal areas, particularly Orissa; sporadic elsewhere; incidence may be higher	Not reported
Tissue schizogony	5-6 days	8 days	9 days	13 days	8-9 days
Erythrocytic phase	48 hours	48 hours	49-50 hours	72 hours	24 hours
Red cells affected	All	Reticulocytes	Reticulocytes	Mature RBC’s	?
Merozoites per tissue schizont	40000	Over 10000	15000	2000	?
Merozoites per red cell schizont	8 – 32	12 – 24	4 – 16	6 – 12	10-16
Relapse from persistent liver forms	No	Yes	Yes	No, but blood forms can persist up to 30 years	No
Fever pattern	Tertian, sub tertian	Tertian	Tertian	Quartan	Quotidian
Severe malaria	Up to 24%	Up to 22%	Very rare	Very rare	6-10%
Drug resistance	Yes	Yes	No	No	No
See Comparison of Four Human Plasmodium species at CDC

Ring Forms of Malaria Parasites on Thin Blood Smear (Courtesy: CDC DPDx Image Library)

Further Reading:

1. Rich SM, Ayala FJ. Evolutionary Origins of Human Malaria Parasites. In Krishna R. Dronamraju, Paolo Arese (Ed). Emerging Infectious Diseases of the 21st Century: Malaria – Genetic and Evolutionary Aspects. Springer US 2006. pp.125-146.
2. Daneshvar C et al. Clinical and Laboratory Features of Human Plasmodium knowlesi Infection. Clinical Infectious Diseases 2009;49:852–860.
3. Tong-Soo Kim et al. Prevalence of Plasmodium vivax VK210 and VK247 subtype in Myanmar. Malaria Journal 2010;9:195. doi:10.1186/1475-2875-9-195. Available at http://www.malariajournal.com/content/9/1/195
4. Rosenberg R, Wirtz RA, Lanar DE, Sattabongkot J, Hall T, Waters AP, Prasittisuk C. Circumsporozoite protein heterogeneity in the human malaria parasite Plasmodium vivax. Science 1989;245:973-976.
5. Han GD, Zhang XJ, Zhang HH, Chen XX, Huang BC. Use of PCR/DNA probes to identify circumsporozoite genotype of Plasmodium vivax in China. Southeast Asian J Trop Med Pub Health 1999;30:20-23.
6. Branquinho MS, Lagos CB, Rocha RM, Natal D, Barata JM, Cochrane AH, Nardin E, Nussenzweig RS, Kloetzel JK. Anophelines in the state of Acre, Brazil, infected with Plasmodium falciparum, P. vivax, the variant P. vivax VK247 and P. malariae. Trans R Soc Trop Med Hyg 1993;87:391-394.
7. Kain KC, Brown AE, Webster HK, Wirtz RA, Keystone JS, Rodriguez MH, Kinahan J, Rowland M, Lanar DE. Circumsporozoite genotyping of global isolates of Plasmodium vivax from dried blood specimens. J Clin Microbiol 1992;30:1863-1866.
8. Kain KC, Wirtz RA, Fernandez I, Franke ED, Rodriguez MH, Lanar DE. Serologic and genetic characterization of Plasmodium vivax from whole blood-impregnated filter paper discs. Am J Trop Med Hyg 1992;46:473-479.
9. Need JT, Wirtz RA, Franke ED, Fernandez R, Carbajal F, Falcon R, San Roman E. Plasmodium vivax VK247 and VK210 circumsporozoite proteins in Anopheles mosquitoes from Andoas, Peru. J Med Entomol 1993;30:597-600.
10. Kain KC, Brown AE, Lanar DE, Ballou WR, Webster HK. Response of Plasmodium vivax variants to chloroquine as determinated by microscopy and quantitative polymerase chain reaction. Am J Trop Med Hyg 1993;49:478-484.
11. González-Cerón L, Rodriguez MH, Nettel JA, Villarreal C, Kain KC, Hernández JE. Differential susceptibility of Anopheles albimanus and Anopheles pseudopunctipennis to infections with coindigenous Plasmodium vivax variants VK210 and VK247 in southern Mexico. Infect Immun 1999;67:410-412.
12. Sutherland CJ et al. Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally. J Infect Dis. 15 May 2010;201(10):1544-1550.[Full Text]
13. Xin-zhuan Su. Human Malaria Parasites: Are We Ready for a New Species? J Infect Dis. 2010;201(10):1453-1454. [Extract]
14. Akpogheneta O. Researchers Identify New Malaria Species. The Faster Times. Available at http://thefastertimes.com/globalpandemics/2010/04/22/researchers-identify-a-new-malaria-species/
15. Oguike MC et al. Plasmodium ovale curtisi and Plasmodium ovale wallikeri circulate simultaneously in African communities.
    Int J Parasitol. 23 Feb 2011 [Pub Med Abstract]
16. David Tordrup et al. Variant Plasmodium ovale isolated from a patient infected in Ghana. Malaria Journal 2011;10:15. doi:10.1186/1475-2875-10-15. Full text at http://www.malariajournal.com/content/10/1/15
17. Characterization of Human Malaria Parasites. Available at http://www.mrcindia.org/MRC_profile/profile2/Characterization of human malaria Parasites.pdf
18. Carter R, Mendis KN. Evolutionary and Historical Aspects of the Burden of Malaria. Clinical Microbiology Reviews. October 2002;15(4):564-594. Full text at http://cmr.asm.org/cgi/content/full/15/4/564
19. Cyrus Daneshvar, Timothy M. E. Davis, Janet Cox-Singh, Mohammad Zakri Rafa’ee, Siti Khatijah Zakaria, Paul C. S. Divis, Balbir Singh. Clinical and Laboratory Features of Human Plasmodium knowlesi Infection. Clinical Infectious Diseases 2009;49:852–860
20. Chaturong Putaporntip, Thongchai Hongsrimuang, Sunee Seethamchai et al. Differential Prevalence of Plasmodium Infections and Cryptic Plasmodium knowlesi Malaria in Humans in Thailand. The Journal of Infectious Diseases 2009;199:1143–1150
21. Balbir Singh, Lee Kim Sung, Anand Radhakrishnan et al. A large focus of naturally acquired Plasmodium knowlesi infections in human beings. The Lancet 2004;363(9414):1017-1024
22. Janet Cox-Singh, Balbir Singh. Knowlesi malaria: newly emergent and of public health importance? Trends in Parasitology. 2008;24(9):406-410
23. Peter Van den Eede, Hong Nguyen Van, Chantal Van Overmeir et al. Human Plasmodium knowlesi infections in young children in central Vietnam. Malaria Journal 2009;8:249. Full Text at http://www.malariajournal.com/content/8/1/249
24. Lee K-S, Divis PCS, Zakaria SK, Matusop A, Julin RA, et al. Plasmodium knowlesi: Reservoir Hosts and Tracking the Emergence in Humans and Macaques. PLoS Pathog 2011;7(4):e1002015. doi:10.1371/journal.ppat.1002015. Available at http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002015
25. Dhangadamajhi G, Kar SK, Ranjit MR. High prevalence and gender bias in distribution of Plasmodium malariae infection in central east-coast India. Tropical Biomedicine 2009;26(3): 326–333. Available at http://www.msptm.org/files/326_-_333_Ranjit_MR.pdf
26. Rajagopalan PK, Pani SP, Das PK, Jambulingam P. Malaria in Koraput district of Orissa. Indian J Pediatr. 1989 May-Jun;56(3):355-64.
27. Ashwani Kumar, Neena Valecha, Tanu Jain, Aditya P. Dash. Burden of Malaria in India: Retrospective and Prospective View. Am. J. Trop. Med. Hyg. 2007;77(6_Suppl):69-78. Full Text at http://www.ajtmh.org/cgi/reprint/77/6_Suppl/69
28. Malaria situation. National Vector Borne Disease control Programme. Available at http://nvbdcp.gov.in/Doc/Malaria%20Situation_Sep.pdf
29. Matteelli A, Castelli F, Caligaris S. Life cycle of malaria parasites. In Carosi G, Castelli F. (Ed) Handbook of Malaria Infection in the Tropics. Associazione Italiana ‘Amici di R Follereau’ Organizzazione per la Cooperazione Sanitaria Internazionale. Bologna. 1997. pp. 17-23. Available at http://www.aifo.it/english/resources/online/books/other/malaria/2-Lifecycle%20of%20malarial%20parasite.pdf
30. Rogerson SJ, Carter R. Severe Vivax Malaria: Newly Recognised or Rediscovered? PLoS Med. 2008;5(6):e136. Full Text at http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0050136
31. Genton B, D’Acremont V, Rare L, Baea K, Reeder JC et al. Plasmodium vivax and Mixed Infections Are Associated with Severe Malaria in Children: A Prospective Cohort Study from Papua New Guinea. PLoS Med 2008;5(6):e127. Full Text at http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0050127
32. Tjitra E, Anstey NM, Sugiarto P, Warikar N, Kenangalem E, et al. Multidrug-Resistant Plasmodium vivax Associated with Severe and Fatal Malaria: A Prospective Study in Papua, Indonesia. PLoS Med 2008;5(6):e128. Full Text at http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0050128

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Human, Parasite, Mosquito Genome

With the turn of the millennium, the genome of humans, the Anopheles mosquito and the P. falciparum parasite have been sequenced. Thus for the first time, a wealth of information is available for all the three species that comprise the life cycle of the malaria parasite and this would help in a better understanding of the interactions among the three species that have long been evolving together. The genome sequence of P. falciparum was published in Nature (Nature, Plasmodium genomics special issue, 3rd October 2002 See http://www.nature.com/nature/malaria/index.html) and of the mosquito Anopheles gambiae was published in the same week in Science (Science, The Mosquito Genome: Anopheles gambiae, 298:5591; 4th October, 2002 See http://nora.embl.de/ivica/publications/12364791.pdf). The human genome sequence was published simultaneously in Nature (Initial sequencing and analysis of the human genome. Nature 409, 860-921 (15 February 2001) Available at http://www.nature.com/nature/journal/v409/n6822/full/409860a0.html ) and Science (Science Special Issue, February 2001 Vol 291, Issue 5507, Pages 1145-1434. Available at http://www.sciencemag.org/content/vol291/issue5507/index.dtl16 ) in February 2001. The sequencing of the genes opens up new approaches to the development of drugs, vaccines, insecticides and insect repellents, as well as intervention into malarial transmission. Genetic tools will also enable sampling of parasite, mosquito, and human genomes in malaria affected areas to support the malaria control activities.

The Malaria genome:

The sequencing of P. falciparum resulted from an international collaboration established in 1996, comprising of NIAID (National Institute of Allergy and Infectious Diseases), the Wellcome Trust, the Burroughs Wellcome Fund and the U.S. Department of Defense. Sequencers worked at The Institute for Genomic Research (TIGR) in Rockville, MD, the Stanford Genome Center in Palo Alto, California, and the Wellcome Trust Sanger Institute in the United Kingdom. The lead investigator, Malcolm Gardner of TIGR, co-authored the Nature paper with 44 researchers working in sites in the United States, the United Kingdom and Australia.

The genome sequence of P. falciparum covers 22.8 million bases of DNA, split into 14 chromosomes. Within the genome, 5279 genes have been identified. Only 733 of the 5279 genes have been identified as enzymes.

Most of the biosynthetic pathways appear to be localized in the apicoplast, a structure within the cell that has its own genome and is similar to the chloroplasts of plants and algae. Although this genome encodes only 57 proteins, it is calculated that around 10 per cent of the proteins encoded by the nucleus may be destined for this structure. The genome sequence also identifies the molecules within the apicoplast that are the targets of several existing drugs, like antibiotics and may open many potential drug targets.

The parasite appears to lack some key biosynthetic pathways; for example, making or interconverting amino acids, making purines, two protein components of ATP synthase (a mitochondrial ATP-producing enzyme) and components of a conventional NADH dehydrogenase complex. It has also been proposed that the regulation of protein levels is controlled through mRNA processing and translation, rather than by gene transcription and this may be another potential drug target.

Regions near the ends of each chromosome of the P. falciparum genome are interesting. The genes residing here encode surface proteins or antigens that are sometimes recognized by the human immune system to stimulate immune response. But exchange of material between chromosome ends gives the parasite a great capacity for change and thereby immune evasion.

The Anopheles genome:

France was the first country to undertake a large-scale sequencing program for the Anopheles genome. As early as 1998, Genoscope and the Unit of Insect Biochemistry and Molecular Biology at the Institut Pasteur sequenced and analyzed the ends of 12,000 large genome fragments from a “bank” set up by Frank Collins at the University of Notre-Dame in the United States. Subsequent work of sequencing the 14,000 genes of the Anopheles was a collective effort of the Anopheles Gambiae Genome Consortium (AGGC) set up in March 2001 and was performed at Genoscope with funds from the French government and at the Celera Genomics Group in Rockville, MD. The strategy selected for the sequencing of the Anopheles’ 280 million bases (Megabases – Mb) was the “whole genome shotgun” method. The institutions contributing to the effort included NIAID, the Special Program of Research on Tropical Diseases of the World Health Organization; European Molecular Biology Laboratory of Germany; the Institute of Molecular Biology and Biotechnology in Crete; the Institut Pasteur in Paris; TIGR; and the universities of Iowa, Rome, Notre Dame, and Texas A&M. Celera’s Robert A. Holt heads a list of 123 authors on the Science paper, submitted on behalf of the AGGC.

Information from the Anopheles genome is giving researchers new insights into mosquito physiology and behavior. Identification of the mosquito genes involved in the parasite’s transmission, resistance to insecticides, the mosquito’s olfactory system, its immunity, its ability to digest blood, its choice of humans as a blood source etc should eventually lead to the development of ways to control the transmission of malaria by this vector.

Some fifty genes are known to be related to the mosquito’s resistance to insecticides and four of these have been identified.

Comparisons between the genome sequence of Drosophila fruit fly (obtained in year 2000) and that of the mosquito have helped in the discovery of the mosquito’s equivalent gene mechanism capable of blocking development of the parasite in the mosquito. This could be a potential target for preventing the development of the parasite.

The mosquito’s smell receptors, are probably implicated in the female Anopheles’ attraction to humans and a whole range of genes associated with smell has been discovered. This will considerably facilitate research on these receptors, and will probably result in the development of new repellents or new attractants. Furthermore, the possibility of a better understanding of the metabolism of the mosquito’s resistance to current insecticides could allow a more ecological use of these products.

Further Reading:

1. Gardner MJ et al. Genome sequence of the human malaria parasite Plasmodium falciparum Available at http://www.nature.com/nature/journal/v419/n6906/full/nature01097.html
2. Holt RA et al. The Genome Sequence of the Malaria Mosquito Anopheles gambiae Science Vol 298; 4 October 2002 Available at http://nora.embl.de/ivica/publications/12364791.pdf
3. The malaria genome unveiled
4. Malaria genomes cracked
5. Sequenced malaria genome exposes novel drug targets
6. http://www.wehi.edu.au/MalDB-www/genomeInfo/MapData/MapData.html
7. MALARIA GENOME DATABASES
8. Plasmodium falciparum Genome Project
9. The malaria genome — and beyond
10. The Mosquito Genome: Anopheles gambiae
11. Genome sequence of the mosquito Anopheles gambiae: the prospects
12. Parasite, Mosquito Genomes Complete Malaria Picture

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