plasmodium knowlesi and diapgnose

E M E R G I N G I N F E C T I O N S I N V I T E D A R T I C L EJames M. Hughes and Mary E. Wilson, Section Editors

Review of Cases With the Emerging Fifth HumanMalaria Parasite, Plasmodium knowlesi

Anu Kantele1,2,3 and T. Sakari Jokiranta3

1Division of Infectious Diseases, Department of Medicine, Helsinki University Central Hospital, 2Department of Medicine, University of Helsinki, and3Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Helsinki, Finland

Human malaria has been known to be caused by 4 Plasmodium species, with Plasmodium falciparum causing

the most-severe disease. Recently, numerous reports have described human malaria caused by a fifth

Plasmodium species, Plasmodium knowlesi, which usually infects macaque monkeys. Hundreds of human cases

have been reported from Malaysia, several cases have been reported in other Southeast Asian countries, and

a few cases have been reported in travelers visiting these areas. Similarly to P. falciparum, P. knowlesi can cause

severe and even fatal cases of disease that are more severe than those caused by the other Plasmodium species.

Polymerase chain reaction is of value for diagnosis because P. knowlesi infection is easily misdiagnosed as less

dangerous Plasmodium malariae infection with conventional microscopy. P. knowlesi infection should be

suspected in patients who are infected with malaria in Southeast Asia. If human–mosquito–human

transmission were to occur, the disease could spread to new areas where the mosquito vectors live, such as

the popular tourist areas in western India.

More than half of the world population lives in malaria

risk areas in Africa, Central and South America, and

South and Southeast Asia, and the annual malaria-

associated mortality approaches 1 million, with 2

children dying from the disease every minute world-

wide (World Malaria report 2009, WHO). For the past

80 years, human malaria has been known to be caused

by 4 Plasmodium species—Plasmodium falciparum,

Plasmodium vivax, Plasmodium ovale, and Plasmodium

malariae—with P. falciparum being responsible for the

most-severe cases. Recently, a fifth Plasmodium species

has been recognized as a cause of malaria in humans.

The newcomer is Plasmodium knowlesi, which was for-

merly known to cause malaria only in macaques.

HISTORY

First described in 1931 in a longtail macaque (Macaca

fascicularis), P. knowlesi was later found to cause natu-

rally acquired malaria in pigtail macaques (Macaca

nemestrina) and the mitred leaf monkey (Presbytis

melalophos), as well. Experimentally, P. knowlesi infects

several other primates, and in 1932, P. knowlesi was

proved to infect humans. Iatrogenic P. knowlesi in-

fection was initially considered to be nonhazardous, and

therefore, the parasite replaced P. vivax as a pyretic agent

in the treatment of neurosyphilis in the early 1930s. This

practice was discontinued, however, as a result of patient

deaths [1].

It was not until 1965 that the first natural infection of

P. knowlesi in humans was reported in an American

traveller returning from Peninsular Malaysia [2]. In

1971, another natural infection was suspected in

Peninsular Malaysia [3]. Despite extensive studies in

Malaysia in the 1960s, no additional reports appeared

until 2004, when Singh et al [4] described 120 cases of

naturally acquired P. knowlesi infection in humans in

Malaysian Borneo. Since then, numerous reports have

been published. By analyzing old blood films, P. knowlesi

Received 9 November 2010; accepted 22 February 2011.Correspondence: Anu Kantele, MD, PhD, Div of Infectious Diseases, Helsinki

University Central Hospital, P.O.Box 348, FIN-00029 HUS, Finland ([email protected]).

Clinical Infectious Diseases 2011;52(11):1356–1362� The Author 2011. Published by Oxford University Press on behalf of the InfectiousDiseases Society of America. All rights reserved. For Permissions, please e-mail:[email protected]/2011/5211-0014$14.00DOI: 10.1093/cid/cir180

1356 d CID 2011:52 (1 June) d EMERGING INFECTIONS

has been shown to have caused malaria in a great number of

patients since 1996 [5].

MONKEY MALARIA

At least 26 Plasmodium species are known to infect primates, yet

natural transmission of a nonhuman Plasmodium species to

humans is rare. The host specificity has been shown to be sur-

prisingly strict: eg, Plasmodium reichenowi causing malaria in

chimpanzees fails to infect humans [6]. Similarly, P. falciparum

causes only mild parasitemia in chimpanzees. These kinds of

differences appear to be caused by species-specific erythrocyte

recognition profiles [6] or different binding of sporozoites to

liver cells [7]. P. knowlesi is clearly not strictly species-specific,

because both experimental monkey-to-human and human-to-

human transmission have proved to be possible [8]. Other

mechanisms of species specificity of plasmodia are attributable

to vector restriction, vector feeding preferences, and vector

species specificity. P. knowlesi transmission is vector restricted in

that the parasite can be transmitted only by certain Anopheles

mosquitoes [1, 9]. At least 2 main vectors in the Anopheles

leucosphyrus group, Anopheles latens and Anopheles cracens,

are known to be forest feeders, biting both humans and mac-

aques at evening or during the night [9, 10]. Taken together,

numerous mechanisms restrict the nonhuman Plasmodium

species from infecting humans, yet P. knowlesi has proved to

make a significant exception to this rule.

In the natural hosts, P. knowlesi causes an asymptomatic low-

grade parasitism or mild disease [4, 10]. It is not known whether

human P. knowlesi infections are obtained only from mosquitoes

fed on macaques or whether natural human-mosquito-human

transmission occurs. The zoonotic nature of the infection is

suggested by the lack of case clustering in Sarawakian human

settlements, where most cases have been described, and by the

fact that most patients have a recent history of working or

dwelling in a forest or forest fringe, which is the environment

characteristic of the vector mosquitoes [10, 11]. During human

infection, gametocytes are formed, but at low levels, which

supports the suggestion that transmission is primarily zoonotic

[12]. Because of the environmental preference of the natural

vector mosquitoes, urban transmission is not likely to occur.

The situation might change if human-mosquito-human trans-

mission occurred, because at least one widely distributed urban

mosquito species (Anopheles stephensi) has experimentally been

shown to be a possible vector for P. knowlesi (reviewed by

Coatney et al [1]).

CASES IN MALAYSIA

In the first study to recognize P. knowlesi as a causative organism

of human malaria in Sarawak, Malaysia, the authors investigated

cases that were identified by microscopic examination as

being due to P. malariae but that had negative polymerase chain

reaction (PCR) results [4]. When new primers were applied,

58% of the cases that originally had PCR results negative for

P. malariae were found to have positive results for P. knowlesi,

resulting in the identification of 120 cases of P. knowlesi

infection in humans.

Thereafter, P. knowlesi was identified in 266 of the samples

from 960 hospitalized Sarawakian patients with malaria [13], in

41 of 49 archived blood-films collected in Sabah [13], and 35 of

47 in Sarawak [5], in Malaysian Borneo, and in a total of 89 cases

in Peninsular Malaysia [9, 13, 14]. In 2009, a prospective report

on P. knowlesi infections in Sarawak was published that con-

tained the clinical picture and the laboratory findings for 107

patients with P. knowlesi infection during 2006–2008 [15]. Since

2004, 5 fatal cases have been reported [13, 16].

CASES IN OTHER SOUTHEAST ASIAN

COUNTRIES

After identifying cases in Malaysia, several patients with

P. knowlesi infection have been documented in other Southeast

Asian countries (Figure 1; Table 2). Diagnosis in all of these cases

was based on PCR analysis, but malaria was detected in prac-

tically all of the cases using microscopy. In 2004, P. knowlesi

malaria was described in a patient who lived in a suburb of

Bangkok, Thailand, and who had visited southern Thailand near

the Myanmar border [27]. Subsequently, P. knowlesi has been

identified in 10 patients from southern and southwestern

Figure 1. Plasmodium knowlesi infections reported in humans inSoutheast Asia (modified from Cox-Singh and Singh [10]). The grayarea indicates the geographic distribution of Anopheles leucosphyrusgroup mosquitoes, which are the main vectors for P. knowlesi. The figuresrepresent numbers of patients with a reported infection due toP. knowlesi. The figures include both local patients and travelersreturning from that area. For 1 traveler, the area in which the infectionwas obtained remained unclear, because he had visited several countries(Thailand, Indonesia, Peninsular Malaysia, and Vietnam) [17].

EMERGING INFECTIONS d CID 2011:52 (1 June) d 1357

Thailand [28]; 33 patients from Myanmar [29, 30]; 5 pa-

tients, including 2 patients with a history of staying in forest

areas, from the Philippines [32]; 2 soldiers and 4 other patients

from northwestern Singapore [24–26]; 5 patients from central

Vietnam [31]; and 1 patient, a gold miner from southern

Borneo, in Indonesia [21]. As Sulistyaningsih et al [21] have

concluded, it appears to be possible that the parasite is widely

distributed throughout Borneo.

CASES IN TRAVELERS

After the first reported case of P. knowlesi infection in a traveler

(Table 2) in 1965 [2], the next case was reported 42 years later. In

2007, a Finnish man was infected during a 4-week visit to

Peninsular Malaysia, where he stayed in a jungle area for 5 days

[23]. He did not take any malaria prophylaxis, and his illness

was originally diagnosed by microscopy as coinfection due to

P. falciparum and P. malariae. This and the next few cases were

confirmed using PCR and sequencing or P. knowlesi–specific

nested PCR. The third reported case was in a Swedish man who

had been travelling in Malaysian Borneo for 2 weeks and stayed

in a jungle for 1 week. Rapid diagnostic test results for malaria

was negative, but microscopic examination revealed low para-

sitemia with a suspicion of P. malariae infection [18]. The

fourth report described a woman from the United States who

had visited the Philippines, her native country [33]. The fifth

case was in a Malaysian immigrant in the Netherlands [19]. The

sixth traveller documented with P. knowlesi infection was an

Australian man working 10 days a month near woodlands in

Indonesian Borneo in the province located farthest from the

Malaysian border [22]. The seventh case was in a Spanish man

who had returned from a long tour in Southeast Asia [17]. In

addition to these reports, the British health statistics list 1 case

of P. knowlesi infection in 2006 in a patient who had visited

Brunei [20].

CLINICAL PICTURE AND OUTCOME

The clinical picture and outcome of P. knowlesi infection is

described in a prospective study involving 107 patients in the-

Sarawak hospital in Malaysia during the period 2006–2008

[15]. The clinical and laboratory profiles of these patients

resembled those of patients infected with other Plasmodium

species. A Vietnamese report suggests that asymptomatic in-

fections may also occur [34]. In Malaysia, the patients with

P. knowlesi infection typically presented with a nonspecific

febrile illness with daily fever and chills. Other frequent symp-

toms included headache, rigors, malaise, myalgia, abdominal

pain, breathlessness, and productive cough. Tachypnea, pyrexia,

and tachycardia were common clinical signs. All patients had

thrombocytopenia at hospital admission or on the following

day, yet none of the patients had clinical coagulopathy.

At hospital admission, only a few of the patients had anemia,

whereas mild hepatic dysfunction was relatively common.

The great majority of patients (94%) experienced no compli-

cations, and the infection responded well to chloroquine and

primaquine treatment [15].

Applying the World Health Organization criteria for

P. falciparum infection, P. knowlesi infection was evaluated

as severe in 7% of the cases, with the most frequent comp-

lication being respiratory distress with pulmonary rather than

metabolic etiology. A strong correlation was found between

parasite density and the development of respiratory distress.

Parasite density was also strongly and independently associated

with renal dysfunction. Two of the patients died, representing

a case fatality rate of 1.8% [15]. Phylogenically, P. knowlesi

is relatively closely related with P. vivax; therefore, it is not

surprising that infections with these 2 species share some com-

mon features, such as occasional severity and marked throm-

bocytopenia [35]. However, disease caused by P. knowlesi is

somewhat more severe than disease caused by P. vivax, although

also vivax malaria can be severe in a significant number of cases,

at least in certain regions (severe malaria in �3%, compared

with 7% in individuals with P. knowlesi malaria) [15, 35].

In P. knowlesi infections, neurological symptoms are rare,

and no cases of cerebral malaria have been reported [15]. A

post-mortem study of a lethal P. knowlesi case suggests, however,

that P. knowlesi–infected red cells can sequester in capillaries

of the brain, heart, and kidneys [16].

Similarly to P. falciparum, P. knowlesi can cause severe or even

fatal disease. It has a short lifecycle of 24 h [1], enabling a rapid

progression of the disease (Table 1). The erythrocyte invasion by

P. knowlesi is not restricted to young or old cells, which allows

high parasitemia, and development of the parasite in eryth-

rocytes is asynchronous. The threshold for hyperparasitemia in

P. knowlesi infections is lower than in P. falciparum malaria

and, at least theoretically, hyperparasitemia, together with the

shorter asexual cycling time, may even render P. knowlesi more

virulent in its severe form. In 3 of the 5 reported lethal cases,

a high parasitemia was seen (15%, .10%, and .10 parasites per

high-power microscope field) [13, 16]. Clinicians treating these

patients should be aware of the potential lethal outcome of the

infection.

TREATMENT

As malaria due to P. knowlesi might progress rapidly into severe

disease, it should be treated like P. falciparum malaria if the

species identification is based on microscopic examination alone

or if coinfection with P. falciparum cannot be excluded with

certainty using PCR. P. knowlesi itself appears to be susceptible

to numerous alternative treatments (Table 1). The majority of


Malaysian patients were primarily treated with chloroquine

[4, 15, 36], the Finnish patient was treated with quinine

and doxycycline [23], the Swedish patient was treated with

mefloquine [18], and the American patient was treated with

the combination of atovaquone and proguanil [33]. This sug-

gests that P. knowlesi malaria can be treated with all of these

agents (Table 1). In general, antimalarial medication should

be commenced at once after patients are found to have positive

blood smear results, and the species can be identified or con-

firmed later. In P. knowlesi infection, commencing medication

immediately is as important as it is in P. falciparum infection

because of the potentially severe nature of both infections and

because of the difficulty in distinguishing between these 2 species

or determining whether there is a mixed infection on the basis

of microscopic examination alone.

Two plasmodium species, P. vivax and P. ovale, may become

dormant as liver hypnozoites and can cause one or more re-

lapses of disease even years after the original infection if

primaquine is not given to eradicate this form of the parasite

(Table 1). After the primary treatment, Malaysian patients

have been treated with primaquine to eliminate possible hyp-

nozoites [15, 36]; however, there is no evidence from either

macaque or human infections on relapses or indicating that that

hypnozoites exist in P. knowlesi infection. Therefore, it appears

to be unlikely that primaquine administration is beneficial or

necessary in P. knowlesi infection.

Table 1. The 5 Plasmodium-Species Causing Malaria in Humans

Variable

Plasmodium

falciparum

Plasmodium

knowlesi

Plasmodium

vivax

Plasmodium

ovale

Plasmodium

malariae

Main site ofdistribution,proportion inthe endemicarea (%)

Global,most commonin Africa,80–90 (Africa)

Malaysia (andneighboringareas), 1–60

Mostly in Asia, 50–80 Africa, 5–8 Global, 0.5–3

Life threatening yes yes yes no no

Red cellsand fevercycle, days

2 1 2 2 3

Infectedred cells

All All Young cells Young cells Old cells

Parasitemia Can be high Can be high ,2% ,2% ,2%

Liverhypnozoites

No Probably no Yes Yes No

Treatmenta Combinations witharthemisininederivatives

Atovaquoneplus proguanil

Mefloquine

Quinine 6 doxycycline

Probably allthe medicationslisted for theother plasmodia

Chloroquinefollowed byprimaquine

Chloroquinefollowed byprimaquine

Chloroquine

Medicalprevention


DoxycyclineMefloquine

Not known,probably


Chloroquine

Doxycycline

Mefloquine


Chloroquine(resistance rare)

Doxycycline

Mefloquine

No preventingmedicationcan preventliver hypnozoites


Chloroquine

Doxycycline

Mefloquine

No preventingmedicationcan preventliver hypnozoites


Chloroquine

Doxycycline

Mefloquine

a If a patient with a P. falciparum infection has high parasitemia, poor condition, and/or complications, intravenous medication (artesunate or quinine with or

without doxycycline) is recommended. This probably applies also to P. knowlesi infections requiring intravenous medication. Possible use of chloroquine has been

suggested as well [36], but this needs to be further explored before a recommendation can be made.


DIAGNOSIS

Malaria diagnosis is traditionally based on light microscopy

of thin and thick blood smears. In experienced laboratories,

microscopy is reliable, but even in such laboratories, its sensi-

tivity is always not optimal [37]. In P. knowlesi infections,

microscopic examination has been used to identify plasmodia

in the samples and to determine the level of parasitemia.

Microscopic examination, however, fails to correctly identify

the species of P. knowlesi, because its early trophozoites resemble

the ring forms of P. falciparum, and its later stages mimic those

of P. malariae (Figure 2) [8]. This explains why P. knowlesi

infection is usually misidentified as P. falciparum and/or

P. malariae infection [2, 5, 12, 13, 23, 25, 28]. Because

P. knowlesi malaria is life threatening, this parasite should

always be suspected in cases in which microscopic examination

suggests P. malariae, but in which the patient has either severe

disease, considerable parasitemia (.0.1%; ie, a parasite count

of .5000 parasites/lL), or a recent history of visiting woods

or their vicinity in Southeast Asia [5].

The PCR techniques that are used for the diagnosis of malaria

and are not specifically designed for detection of P. knowlesi

fail to detect it [17, 22, 38]. This parasite has been detected only

with P. knowlesi–specific PCR assays that use nested PCR [4, 28],

real-time PCR [38], or PCR combined with sequencing [23].

The widely used nested PCR assay (using the primers Pmk8 and

Pmk9r), however, occasionally gives false-positive results in ei-

ther uninfected or P. vivax–infected individuals [21, 31, 39],

indicating that a molecular method to test all 5 human malaria

species or verification of identification by sequencing of the PCR

product is needed [21, 28, 31]. A more specific set of primers has

also been suggested [38, 39]. Although PCR analyses can be

done by scraping material from microscopy slides, ethyl-

enediaminetetraacetic acid-anticoagulated blood samples should

be used to minimize contamination.

In several countries, traditional microscopy-based malaria

diagnostics have recently been combined with, or at least par-

tially replaced by, the use of malaria rapid diagnostic tests

(RDTs). Some of the kits available are based on detection of

plasmodium-specific lactate dehydrogenase, whereas others

detect histidine-rich protein II (HRP-2) of P. falciparum either

alone or in combination with a pan-malarial antigen, aldolase.

Currently, no commercially available RDTs are designed

to specifically detect P. knowlesi. Performance of these mainly

P. falciparum– and P. vivax–targeted RDTs in P. knowlesi in-

fection has been reported in a few cases [18, 25], which suggests

that anti-HRP-2 antibodies do not always recognize P. knowlesi

[18], whereas anti-aldolase and anti-LDH antibodies are more

reliable [19, 40]. In conclusion, these tests are not recommended

because of the unreliable results and low sensitivity in detecting

P. knowlesi. Because knowlesi malaria can be symptomatic in

patients with low parasitaemia, RDTs should detect it with

sensitivity of 1 or even ,1 parasite/lL of blood.

PREVENTION OF P. KNOWLESI–INFECTION

As with the other types of human malaria, prevention of

P. knowlesi infection is based on avoiding mosquito bites and

taking preventive medication when indicated. The efficacy of

these measures against P. knowlesi has not been shown, but it

can be assumed that its vector behaves like the vectors of

other Anopheles species, and general precautions for avoiding

the bites of Anopheles mosquitoes probably apply. Current

indoor control measures for malaria do not, however, prevent

zoonotic transmission of malaria by vectors that mainly feed in

the forest [9, 10]. Zoonotic P. knowlesi infection can, accord-

ingly, continue to be a problem for malaria control, and also

poses a significant threat to the renewed efforts aimed at fully

eradicating malaria.

None of the tourists with reported P. knowlesi infection used

preventive medication [18, 19, 22, 23, 33]. Because chloroquine

[4, 15], doxycycline [23], mefloquine [18], or atovaquone-

proguanil in combination [22] have been successfully used in

treatment of the disease, all of these agents could probably be

used as preventive medicines against P. knowlesi, as well.

Figure 2. Examples of challenges in species identification usingmicroscopy. On the left are photos from thin smears from a Finnish touristwith a Plasmodium knowlesi infection after a visit to western PeninsularMalaysia. In the middle are thin smears from patients with Plasmodiumfalciparum infection, and on the right are thin smears from patients withPlasmodium malariae infection (Giemsa or May-Grunwald-Giemsa-staining; original magnification, 31000).


SIGNIFICANCE AND FUTURE PROSPECTS

As the number of human P. knowlesi cases increases, clinicians

and laboratory personnel should be alerted to this emerging—

and potentially lethal—cause of malaria [13, 16]. Laboratory

personnel have thus far only been trained to identify the 4

traditional Plasmodium species. Numerous cases in several

countries may have been misidentified as being due to P. malariae.

Moreover, some malaria RDTs have failed to detect P. knowlesi,

and all RDTs fail to identify the species [18,19]. Therefore,

if rapid and reliable molecular diagnostic methods are not avail-

able to establish the diagnosis of P. knowlesi infection, ei-

ther ordinary malaria microscopic examination or at least

superficial microscopic examination together with RDTs appears

to be necessary to diagnose malaria in general in patients infected

with P. knowlesi.

So far, human P. knowlesi infections have been described in

high numbers only in Malaysia. The great majority are from

Malaysian Borneo [3, 4, 13, 15], yet numerous cases have also

been described in Peninsular Malaysia [9, 13, 14, 23]. Due to the

reports of P. knowlesi malaria obtained in the neighboring

countries, Thailand [27, 28], Singapore [24–26], Brunei [20],

Indonesia [21, 22], Myanmar [29, 30], Vietnam [31], and the

Philippines [32, 33], it appears that P. knowlesi is a natural

parasite of macaques throughout the Southeast Asian region.

The wide distribution of human cases shows that P. knowlesi is

generally able to infect humans, which is also suggested by the

genetic polymorphism of the P. knowlesi identified from human

samples [28]. The fact that P. knowlesi is being identified in

increasing numbers now may be attributable in some areas to

the reduction of cases of P. falciparum and P. vivax infection,

whereas the number of cases of P. knowlesi infection remains

constant. The availability and use of specific PCR methods to

detect P. knowlesi may generate more reports of P. knowlesi

infection, and therefore, the increase in the number of reports is

not necessarily linked to the actual spread of the parasite but

to the use of more-accurate diagnostic methods.

The high number of human cases (Table 2) suggests that

P. knowlesi may be more capable of infecting humans than are

the other species that cause nonhuman primate malaria. To

date, only experimental, and not natural, human-mosquito-

human transmission has been reported [8]. If natural human-

mosquito-human transmission occurred, P. knowlesi could

spread more widely in Asia. This is made possible by the wide

distribution of at least one vector species, Anopheles latens, in

Southeast Asia and in the southern parts of the Indian sub-

continent, including the popular tourist areas in western India

[10] (Figure 1). It remains to be seen whether this kind

of spreading has occurred in the past or will occur in the future,

but to date, neither human-mosquito-human transmission nor

spreading of P. knowlesi has been documented.

Acknowledgments

Potential conflicts of interest. All authors: no conflicts.

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Local

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Indonesia/Borneo 1 1 [21, 22]

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Singapore 6 [24–26]

Thailand 11 [27, 28]

Myanmar 33 [29, 30]

Vietnam 5 [31]

Philippines 5 1 [32, 33]

Total 720 8a

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