Plasmodium knowlesi

Plasmodium knowlesi is a primate malaria parasite commonly found in Southeast Asia.^[1] It causes malaria in long-tailed macaques (Macaca fascicularis), but it may also infect humans, either naturally or artificially.

Plasmodium knowlesi is the sixth major human malaria parasite (following the division of Plasmodium ovale into 2 subspecies). It may cause severe malaria as indicated by its asexual erythrocytic cycle of about 24 hours,^[2][3][4] with an associated fever that typically occurs at the same frequency (i.e. the fever is quotidian).^[2] This is an emerging infection that was reported for the first time in humans in 1965.^[2] It accounts for up to 70%^[5] of malaria cases in certain areas in South East Asia where it is mostly found. This parasite is transmitted by the bite of an Anopheles mosquito.^[5] Plasmodium knowlesi has health, social and economic consequences for the regions affected by it.

History of discovery

The first person to see P. knowlesi was probably the Italian Giuseppe Franchini in 1927 when he was examining the blood of Macaca fascicularis and he noted that it differed from Plasmodium cynomolgi and Plasmodium inui.^[6] It was later seen by Campbell in 1931 in a long-tailed macaque imported from Singapore to the Calcutta School of Tropical Medicine and Hygiene in India. Campbell was interested in kala azar and was working under Napier. Napier inoculated the strain into three monkeys, one of which was a rhesus macaque (Macaca mulatta), which developed a fulminating infection. Knowing that the Protozoological Department were looking for a monkey malaria strain, they handed the original infected monkey to Biraj Mohan Das Gupta, who was the assistant of Robert Knowles. Dr Das Gupta maintained the species by serial passage in monkeys until Dr Knowles returned from leave. In 1932, Knowles and Das Gupta described the species in detail for the first time and showed that it could be transmitted to man by blood passage, but failed to name it. It was named by Sinton and Mulligan in 1932 after Dr Knowles. From early in the 1930s to 1955, P. knowlesi was used as a pyretic agent for the treatment of patients with neurosyphillis.^[7]

In 1957, it was suggested by Garnham et al.^[8] that P. knowlesi could be the fifth species capable of causing endemic malaria in humans.

In 1965, the first case of a naturally occurring infection of knowlesi malaria in humans was reported in an American man who had returned after working in the jungle in peninsular Malaysia.^[9] Although the infecting parasite was initially identified as P. falciparum, one day later it was then identified as P. malariae and it was only confirmed to be P. knowlesi after infected blood was used to inoculate rhesus monkeys.^[7] A second report emerged in 1971 about the natural infection of a man in Malaysia with Plasmodium knowlesi followed by the description of a large focus of human infections in the Kapit Division of Sarawak, Malaysian Borneo.^[7] This was made possible due to the development of molecular detection assays which could differentiate between Plasmodium knowlesi and the morphologically similar Plasmodium malariae. Since 2004, there has been an increasing number of reports of the incidence of P. knowlesi among humans in various countries in South East Asia, including Malaysia, Thailand, Singapore, the Philippines, Vietnam, Myanmar and Indonesia.^[10]

Work with archival samples has shown that infection with this parasite has occurred in Malaysia at least since the 1990s^[11] and it is now known to cause 70% of the malaria cases in certain areas of Sarawak.^[12]

Evolution

Based on a Bayesian coalescent approach the most probable time of evolution of P. knowelsi is 257,000 years ago (95% range 98,000–478,000).^[13] Yakob and coauthors calculated the likelihood of natural host switching from the long-tailed macaque monkey to humans using an evolutionary invasion analysis and demonstrated how this switch was contingent on relative host densities and individual-level mosquito feeding preferences.^[14]

Life cycle

Plasmodium knowlesi parasite replicates and completes its blood stage cycle in 24-hour cycles^[4] resulting in fairly high loads of parasite densities in a very short period of time. This makes it a potentially very severe disease if it remains untreated. Life cycle: merozoite → trophozoites → schizont → merozoite. These stages of Plasmodium knowlesi are microscopically indistinguishable from Plasmodium malariae and the early trophozoites are identical to those of Plasmodium falciparum .

Mosquito stages:^[15] A mosquito ingests gametocytes, which have been formed in the mammalian host. These are either microgametocytes (which are male gametocytes) or macrogametocytes (which are female gametetocytes). These gametocytes mature into microgametes and macrogametes respectively, and then fertilize to form zygotes within the midgut of the mosquito. The zygotes mature into ookinetes, then into oocysts. Finally, the oocysts mature to release sporozoites which move to salivary gland of the mosquito.

Summary: gametocyte → (microgamete or macrogamete) → zygote → ookinete → oocyst → sporozoites.

In man: exoerythrocytic stage (in the liver):^[15] The sporozoites are injected into humans when the mosquito bites and they travel to the liver through blood stream and undergo asexual reproduction to become merozoites through schizonts in the liver cell. Hypnozoites in the liver has not yet been found.

Summary: sporozoites → schizonts → merozoites.

In man: erythrocytic stage (in the blood):^[15] Merozoites are unleashed into the blood stream to infect erythrocytes constituting one asexual cycle of infection of the erythrocytes. Within the red blood cells some merozoites develop into trophozoites, which in turn mature into schizonts that rupture to release merozoites, while others develop into microgametocytes or macrogametocytes. These gametocytes remain in the blood to be ingested by mosquitoes.

Summary: Merozoite → trophozoite → schizont → merozoites.

Epidemiology

P. knowlesi infection is normally considered a parasite of long-tailed (Macaca fascicularis) and pig-tailed (Macaca nemestrina) macaques ^[10][16] but humans who work at the forest fringe or enter the rainforest to work are at risk of infection. With the increasing popularity of deforestation and development efforts in South East Asia, many macaques are now coming in close and direct contact with humans.^[10] Hence more and more people who live in the semi-urban areas are being found to be infected with knowlesi malaria. 2,584 cases of this type of malaria were reported in Malaysia in 2014.^[17]

This parasite is mostly found in South East Asian countries particularly in Borneo, Cambodia,^[18] Malaysia, Myanmar, Philippines, Singapore,^[19] Thailand^[20] and neighboring countries and it appears to occur in regions that are reportedly free of the other four types of human malaria. Infective mosquitoes are restricted to the forest areas. Non-infective mosquitoes are typically found in the urban areas but transmission may occur due to the abundance of mosquitoes in this region.^[2][21] particularly Malaysia,^[7] but there are also reports on the Thai-Burmese border.^[3] One fifth of the cases of malaria diagnosed in Sarawak, Malaysian Borneo are due to P. knowlesi.^[7]

Plasmodium knowlesi is absent in Africa. This may be because there are neither long-tailed nor pig-tailed macaques (the reservoir hosts of P. knowlesi) in Africa, and many West Africans lack the Duffy antigen - a protein on the surface of the red blood cell that the parasite uses to invade.

P. knowlesi is the most common cause of malaria in childhood in the Kudat district of Sabah, Malaysia.^[22]

Vectors

Theoretically there are four modes of transmission: from an infected mosquito to another monkey, from an infected monkey to a human, from an infected human to another human and from an infected human back to a monkey.^[10] In practice human malaria appears to be almost entirely due to monkey to human transmission.

The known vectors belong to the genus Anopheles, subgenus Cellia, series Neomyzomyia and group Leucosphyrus.^[4] Mosquitoes of this group are typically found in forest areas in South East Asia but with a greater clearing of forest areas for farmland, humans are increasingly becoming exposed to these vectors.

Within the monkey population in Peninsular Malaysia, Anopheles hackeri is believed to be the main vector of P. knowlesi: although A. hackeri is capable of transmitting malaria to humans,^[23] it is not normally attracted to humans and seems unlikely to be an important vector for transmission to humans.^[24]

Anopheles latens is attracted to both macaques and humans and has been shown to be the main vector transmitting P. knowlesi to humans in the Kapit Division of Sarawak, Malaysian Borneo.^[25]

Anopheles cracens has also been reported as a vector of P. knowlesi. Both species of mosquitoes have been shown to contain as many as 1,000 sporozoites suggesting that they may be efficient vectors.^[10]

A study of potential vectors in Malayasia suggests that Anopheles cracens may be an important vector of P. knowlesi.^[26]

Clinical

Two possible modes of transmission to humans have been proposed: either from an infected monkey to a human or from an infected human to another human.

Symptoms typically begin approximately 11 days after an infected mosquito has bitten a person and the parasites can be seen in the blood between 10 – 12 days after infection.^[27] The parasite may multiply rapidly resulting in very high parasite densities that may be fatal.^[27]

Although the current infection rate with Plasmodium knowlesi is relatively low, one risk it presents is misdiagnosis with other forms of malarial parasites such as P. malariae especially when microscopy is used. P. knowlesi can only be accurately distinguished from P. malariae using PCR assay and/or molecular characterization.

Symptoms of P. knowlesi in humans include headache, fever, chills and cold sweats.^[27] Singh et al. (2004)^[7] showed clinical symptoms in 94 patients with single species P. knowlesi infection at Kapit Hospital, Sarawak, Malaysian Borneo. Symptoms included fever, chills, and rigor in 100% of patients, headache in 32%, cough in 18%, vomiting in 16%, nausea in 6%, and diarrhea in 4%. Asexual cycle of the parasite in humans and its natural host macaque is about 24 hours.^[2][3][4] Hence the disease may be called quotidian malaria,^[2] in concert with designation of tertian malaria and quartan malaria.^[15] In addition to a lab diagnosis using PCR assay, knowlesi malaria may also present itself with elevated levels of C-reactive protein and thrombocytopenia.

This parasite causes non-relapsing malaria^[28] due to lack of hypnozoites in its exoerythrocytic stage.^[29]

While infection with this organism is normally not serious, life-threatening complications or even death may occur in a minority of cases. The most common complications are respiratory distress, abnormal liver function including jaundice and renal failure. Mortality in one series of cases was about 2%.^[12]

Diagnosis

P. knowlesi infection is diagnosed by examining thick and thin blood films in the same way as other malarias. The appearance of P. knowlesi is similar to that of P. malariae and is unlikely to be correctly diagnosed except by using molecular detection assays^[7] in a malaria reference laboratory.

The morphology of Plasmodium knowlesi is similar to that of Plasmodium malariae. P. malariae is characterized by a compact parasite (all stages) and does not alter the host erythrocyte's shape or size or cause enlargement. Elongated trophozoites stretching across the erythrocyte, called band forms, are sometimes observed. Schizonts will typically have 8-10 merozoites that are often arranged in a rosette pattern with a clump of pigment in the center.^[30]

Rapid diagnostic tests kits may or may not recognize P. knowlesi because of their specificity.

Currently PCR assay and molecular characterization are the most reliable methods for detecting and diagnosing P. knowlesi infection. PCR identifies the parasite DNA but this technique is not rapid and cannot be used for routine identification. PCR is also expensive and requires very specialized equipment.^[7]

Treatment

Because P. knowlesi takes only 24 hours to complete its erythrocytic cycle, it can rapidly result in very high levels of parasitemia with fatal consequences. Anyone with a severe and rapidly deteriorating condition should be treated aggressively and urgently as if were infected with falciparum malaria.^[4] P. knowlesi responds well to treatment with chloroquine and primaquine. In a clinical study of treatment where response was observed after oral chloroquine was given for three days, and at 24 hours oral primaquine was administered for two consecutive days.,^[31] it was found that this regime gave a rapid response with a median time to parasite clearance of three hours. This was more rapid that is found in Plasmodium vivax malaria where the median time to clearance is between six and seven hours.

Public health, Prevention strategies and Vaccines

1. Mosquito bed nets
2. Medication – Mefloquine, Chloroquine
3. Vector control
4. Residual spraying using insecticides

Pathology

A single post mortem case has been described to date^[32] The patient was a male who became unwell 10 days after exposure. After four days he presented acutely unwell to a hospital. He was found to have a raised eosinophil count, to be thrombocytopaenic, hyponatraemic with an elevated blood urea, potassium, lactate dehydrogenase and amino transferase values. Dengue fever was suspected but ruled out on investigation. Malarial parasites were seen on the blood film and later identified as Plasmodium knowlesi by PCR. At post mortem the liver and spleen were enlarged. The brain and endocardium showed multiple petechial haemorrhages. The lungs had features consistent with acute respiratory distress syndrome. Histological examination showed sequestration of pigmented parasitized red blood cells in the vessels of the cerebrum, cerebellum, heart and kidney without evidence of chronic inflammatory reaction in the brain or any other organ examined. The spleen and liver had abundant pigment containing macrophages and parasitized red blood cells. The kidney had evidence of acute tubular necrosis. Endothelial cells in heart sections were prominent. Brain sections were negative for intracellular adhesion molecule-1.

The overall post mortem picture was very similar to that found in cases of Plasmodium falciparum. There were important differences including the absence of coma despite petechial haemorrhages and parasite sequestration in the brain.

Notes

There are at least two subspecies of P. knowlesi known - P. knowlesi edesoni and P. knowlesi knowlesi. The subspecies P. knowlesi edesoni was described by Garnham in 1963.^[33] It was named after the parasitologist J F B Edeson. It is not known if there are any clinical differences between these two subspecies.

Useful web links

• 1. CDC: cdc.gov/malaria, http://www.cdc.gov/EID/content/14/11/1750.htm
• 2. WHO: http://www.who.int/topics/malaria/en/
• 3. Short film on the discovery of the role of P. knowlesi in human malaria: http://www.abc.net.au/catalyst/stories/2533454.htm

Plasmodium knowlesi genome data

• GeneDB Plasmodium knowlesi

References

Further reading

• Collins, W. E. (2012). "Plasmodium knowlesi: A Malaria Parasite of Monkeys and Humans*". Annual Review of Entomology. 57: 107–121. doi:10.1146/annurev-ento-121510-133540. PMID 22149265.