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Plasmodium knowlesi: from Malaysia, a novel health care threatPlasmodium knowlesi: dalla Malesia una nuova minaccia sanitaria

Sergio Sabbatani1, Sirio Fiorino2, Roberto Manfredi1

1Infectious Diseases, University of Bologna, S. Orsola-Malpighi Hospital, Bologna, Italy;2Department of Internal Medicine, Budrio General Hospital, Budrio, Italy

Until recently there were only four Plas-modium parasites involved in causinghuman malaria: Plasmodium vivax, Plas-

modium malariae, and Plasmodium ovale probablyevolved in ancient times alongside humans (so-called co-evolution), while Plasmodium falci-parum is a proportionally more recent humanparasite, which was passed by monkeys to hu-mans, probably between the late Mesolithic andthe early Neolithic era [1-7]. Monkeys areknown to become infected by over 20 species ofPlasmodium, although the primate-humantransmission of these parasites has been consid-ered since recently an infrequent event, with areduced public health impact. The transmissionof these Plasmodium species has been docu-mented, although with a varied frequency, pre-dominantly in South-East Asia, China, andCentral America [8].The expansion of human activities in former,poorly inhabited areas (i.e. historical rainforests which were turned into productive ar-eas), and woodland regions, which are increas-ingly visited and explored by tourists ontrekking holidays, represent objective elementswhich lead to an increased risk of plasmodialinfection of simian origin. Moreover, the pre-sent demographic expansion of indigenouspopulations in different regions of SouthernAsia and Southern America, also includingwoodland regions or areas close to rain forest,in small villages where primary agriculturaland livestock breeding are performed, repre-sent another, cosiderable epidemiological riskfactor. In this context, there is an overlap of hu-man and simian habitat, in the presence of mos-quitoes belonging to the genus Anopheles,

which are indispensable for human transmis-sion of malaria infection. Among the 20 Plasmodium species which areable to infect monkeys, five have been docu-mented as agents which are infectious for hu-mans: i.e. Plasmodium simium, Plasmodiumbrazilianum (in Southern America), and Plas-modium cynomolgi, Plasmodium inui, and Plas-modium knowlesi (in South-Eastern Asia) [8].Most strains of simian malaria are responsiblefor a mild to moderate, and frequently self-lim-iting, disease in humans; these infections infre-quently require anti-malarial therapy, sincethey are commonly missed. The identificationof P. knowlesi in the states of Sarawak(Malaysian Borneo), and Sabah (another statebelonging to the Malaysia Confederation), be-tween the end of the 20th century and the early21st century, all of them confirmed by molecu-lar biology techniques, has drawn the attentionof researchers and clinicians who operate inSouth-East Asia, and more recently internation-al health care authorities as well, focusing onpatients who experienced typical malaria signsand symptoms, and received in particular a mi-crobiological diagnosis of P. malariae infection[8-10]. A notable feature of these patients is giv-en by frequent, elevated parasitaemia (Figure1), and the severity of the clinical picture,should patients not receive appropriate andtimely treatment [9, 10]. We recall that four fa-talities have also been mentioned in the pub-lished reports [8-10].After identifying bio-molecular primers specif-ic for P. knowlesi, allowed to pose a definitivediagnosis also in subjects who were parasitized,and with their erythrocytes were morphologi-

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cally indistinguishable from those of individu-als infected by P. malariae, but when assessedwith molecular biology primers specific for P.malariae, failed to test positive.The most severe clinical consequence of P.knowlesi malaria is related to the evidence of itsdaily replication cycle and, when not counteredby an adequate therapy, it may rapidly reachpotentially lethal levels of parasitaemia, whilewe recall that the P. malariae cycle has its repli-cation every three days (quartan fever), andnever reaches very elevated levels of para-sitized erythrocytes [9-11]. A particular featureof P. knowlesi is also a similar shape of earlytrophozoite forms to those of Plasmodium falci-parum, whereas all other stages recall the aspectof P. malariae parasites.Recent entomological studies show that themost efficient carrier of human transmission isthe Anopheles leucosphyrus, and the most elevat-ed sting rate is registered when humans are lo-cated close to rain forest (6.74%), while it is low-er inside the forest (1.85%), and it is very low inlonghouses (0.28%), which are the typicalMalaysian dwellings (Figure 2) [12].According to the reported data, it has beenspeculated that humans acquire the infectioninside the forest, when going hunting, and/orwhen coming back to the farm at sunset, afterworking [13]. The monkeys which are the reser-voirs of infection are Macaca nemestina (Figure

3) and Macaca fascicularis (long-tail macaquesand pig-like tail macaques); there are still manydoubts regarding the role of Saimiri scirea as apossible natural reservoir1.The monkey called Macaca mulatta, which doesnot have a Malaysian origin, may become inci-dentally infected by P. knowlesi, like humans.When the latter monkey is infected with P.knowlesi, it becomes sick, and may die. This isthe reason why this species may represent a re-liable animal study model2. In its natural hosts,P. knowlesi causes an asymptomatic, low gradeparasitaemia, which is in clear contrast with ahyperparasitaemia followed by death in thewidely used experimental model of the Rhesusmonkey (i.e. Macaca mulatta). As a conse-quence, the general appraisal is that humansacquire the infection in the forest when huntingor when coming back to the farm at the sunset,after work, while the carrier seems to acquirethe Plasmodium from wild monkeys; this opin-ion is supported by the absence of evidence ofepidemic clusters inside longhouses (untilnow), and the highest number of infected mos-quitoes has been found in the rain forest, or atits margins [13].After the early reports of Malaysian malaria fo-ci, involving the native population, and identi-fication of anecdotal imported cases in Euro-pean or Northern American tourists who spenta short time inside the rain forest, or close to

1Professor Babir Singh of Malaysia-Sarawak University believes that the Saimiri scireamonkey may be used as an experimental mod-el of infection rather than a natural model of infection (B. Singh, Personal Communication, 2010).2At present, the Macaca mulatta (Rhesus monkeys) is fairly common in Western Afghanistan, in India, in Northern Thailand; in addi-tion, it was historically abundant present in Southern China and Tibet, although hunting caused its sharp decline.

Figure 1 - A thin blood smear stained with May-Gr�nwald Giemsa, from a fatal human case of Pla-smodium knowlesi infection. Figure 2 - A longhouse, the typical Malaysian house.

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South-East forests, the researchers’ attentionshifted towards P. knowlesi [14-18]. In the year 2008, the journal Nature first pub-lished the nuclear genomic sequence of P.knowlesi [19]. It was the first complete “se-quencing” of a simian plasmodium, which alsomakes a comparison possible with the genomicsequence of Plasmodium vivax, and that of otheralready sequenced plasmodia. As opposed toother plasmodium genomic patterns, familiarputative antigenic variants are scattered due tothe genomic sequence, and they are associatedto repeated intrachromosomal telomeres. Someof these genomic families, the so-called KIRs,contain sequences which are related to one halfof the intracellular domain CD99 of the animalhost, so that it was supposed that these se-quences may lead to an unusual form of molec-ular mimicry [19, 20].General, relevant information has been provid-ed by the recent observations and by epidemi-ological surveys conducted in Malaysia. Thehuman habitat is increasingly overlapping thatof primates. This phenomenon is mainly due tothe expansion of human-driven activities (agri-culture, tree harvesting aiming to provide finewood, and livestock farming), in areas wheremonkeys do live. The second emerging aspect is related to the in-creased presence and activity (i.e. trekking) ofWestern tourists, who after travelling in theseinhospitable areas for a limited period of time,

return to their countries of origin [11]. In thiscase, the poor knowledge of simian plasmodiaand related diagnostic problems may disorien-tate the physicians, on the basis of a standardmicrobiological-pa rasitological examination,which does not allow P. malariae to be distin-guished from Plasmodium knowlesi, therebyputting the patient’s life at risk [11].Molecular biology techniques prove extremelyuseful in depicting the general epidemiology ofmalaria, and to completely clarify the role andimportance of mixed infections which are stilllargely underestimated [11]. However, this testing is expensive, and not eas-ily accessible to all countries, with special refer-ence to developing nations.When taking into account the epidemiologicalcharacteristics of the areas where epidemic fociand isolated imported cases of a “travel-relat-ed” P. knowlesi disease have been registered, itdoes not seem possible to establish definitivelywhether infection was acquired from an animalreservoir (i.e. Macacus monkeys), or whethercases of interhuman contagion occurred. Any-way, it is now clear that these animal patholo-gies are no longer the expression of episodicfacts, but represent a true health care emer-gency in South-East Asia.Figure 4 gives an overview of the co-evolution-ary pathway of malaria Plasmodia togetherwith that of the genus Homo. From hominids(group A), until contemporary humans (groupC), with the appearance of Plasmodium falci-parum which penetrated this scenario by infect-ing humans (group B) in the Mesolithic and ear-ly Neolithic: these humans live in simple huts atthe edge of the rain forest, close to fields usedfor early agriculture. The early introduction ofthe first human activities helped the spread ofPlasmodium falciparum malaria in the patho-cenosis of prehistoric populations, unavoidablyconditioning economic and anthropological de-velopment.With regard to the appearance of a novel malar-ia parasite which is epidemiologically relevantto humans, the hope is that this novel patho-cenosis remains circumscribed to regions inSouth-East Asia, without the involvement offurther tropical and equatorial areas of othercontinents. In India, Thailand and WesternAfghanistan3, the diffuse presence of Macaca

3We recall that Afghanistan is a country where Western troops are engaged in military activities. Surveillance is also due to the per-sonnel involved.

Figure 3 - A long-tailed macaque (Macaca nemestina).

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mulatta may represent a potential reservoir forP. knowlesi, opening the way to an epidemiolog-ical spread of the infection to regions whichhave not been involved by the problem untilnow. Should this occur, catastrophic effects areexpected, not only for the native populationswho lack a specific immune memory, but alsofor the ever growing number of tourists who,after making their excursions in the rain forest,will be exposed to the risk of a cumbersomeand displacing microbiological examination, atthe time of becoming sick and coming back tothe country of origin. Considerable complications related to the tem-porary lack of commercial, rapid and specificparasitological diagnostic tools, and possiblesubsequent delays in disease recognition andmanagement, are expected in the immediatemean time, since the novel and rapid diagnos-tic kits will become available shortly andworldwide. According to information updated at the end ofNovember 2010, some commercial, rapid diag-nostic kits including also P. knowlesi recombi-nant antigens, are under very advanced devel-opment, and are expected to add significantlyto the prompt and timely recognition of thefifth human Plasmodium, in a route compara-ble to that already traced several years ago forthe rapid laboratory search of other humanmalaria Plasmodia.

As anticipated, diagnostic retardation may be-come fatal, should P. knowlesi infection not beidentified in a reasonably short time. A recent prospective clinical study conductedin the Sarawak regions of Malaysian Borneo,disclosed that approximately 10% of patientsinfected with P. knowlesi had severe signs andsymptoms already upon presentation of brieflylater, and 1-2% of cases have a fatal outcome[21]. The complications occurred in patientswho survived an acute, severe P. knowlesimalaria attack, include the acute respiratorydistress syndrome (ARDS), liver and/or kidneyend-organ dysfunction, hypotension, with orwithout a parasitaemia over 100,000 tropho-zoites/µL. All lethal cases of P. knowlesi disease were char-acterized by prominent abdominal signs andsymptoms, associated to a combined kidney-liver dysfunction, and the concurrent hyperpar-asitaemia [22].From a histopathological point of view, the postmortem lesions observed in a 40-year-old manwho rapidly deceased after 14 days with a “col-lapse”-like disease and quick death, was similarto that observed in the fatal P. falciparum malar-ia. It was the typical sequestration of pigment-ed, parasitized erythrocytes in the cerebellum,brain, heart, and lung vessels without any evi-dence of a chronic inflammatory reaction in thecentral nervous system or other examined or-

Figure 4 - The picture depicts the biological and phylogenetic evolutions of Plasmodium vivax, Plasmodiummalariae and Plasmodium ovale (Group A), compared with the more recent evolution pathway of Plasmodiumfalciparum (Group B). Plasmodium knowlesi (belonging to Group C) is the last Plasmodium species which stea-dily entered the human pathology, and is related to productive and tourist human activities carried out inthe forest regions of Malaysia and Borneo. The image was taken and modified from some studies publishedby Luigi Capasso (quoted among references), especially with Group C malaria parasites [1, 21].

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gans, while an extensive tubular necrosis in-volved the kidneys.The clinical differences, i.e. the absence of comaand other neurological complications notwith-standing the petechial haemorrhage, regardlessof the parasite sequestration inside the brain,are characteristic features [22].Until now, the therapeutic perspectives seemless worrying when prompt diagnosis is ob-tained during the initial disease phases. In avery recent clinical study performed at theKapit Hospital (Sarawak, Malaysian Borneo),the authors assessed the response to a treat-ment with chloroquine at 10 mg/kg of bodyweight, followed by 5 mg/kg after 6, 24, and 48hours), for a total of 25 mg/kg [23]. Twenty-four hours later, after excluding a deficiency ofglucose 6-phosphate dehydrogenase, oral pri-maquine (at 15 mg/kg of body weight) was de-livered for two consecutive days (as a gameto-cidal drug). All patients had a non-severe, non-complicat-ed malaria, with a parasitaemia ≤100,000trophozoites/micron. All treated subjects suc-cessfully benefited from therapy, and 96% of

them had a frank improvement of signs andsymptoms already after the first 24 hours oftreatment [23]. The data presented in thisMalaysian study demonstrate that convention-al chloroquine dosages are associated to an ini-tial parasite clearance which was more rapid,when compared to that of P. vivax (which act-ed as the control group in the Malaysian expe-rience); furthermore, this therapeutic responseproved to be the most rapid, amongst allmalaria forms [24]. These last studies are very encouraging, sincethey demonstrate that a low-cost drug likechloroquine may be affordable in developingcountries [23]. The authors, however, recom-mend to develop further rigorous studies, in or-der to assess whether this treatment may beused also in the severe forms of P. knowlesimalaria.

Key words: malaria, Plasmodium knowlesi, emerg-ing parasite diseases.

Conflicts of interest, funding, sponsorship, acknowl-edgements: none.

Epidemic foci of Plasmodium knowlesi malariahave been identified during the past ten years inMalaysia, in particular in the States of Sarawakand Sabah (Malaysia Borneo), and in the Pahangregion (peninsular Malaysia). Based on a reviewof the available recent international literature, theauthors underline the importance of molecularbiology examinations, polymerase chain reac-tions (PCR), performed with primers specific forP. knowlesi, since the current microscopic exami-nation (haemoscope) may fail to distinguish P.knowlesi from Plasmodium malariae, due to the verysimilar appearance of the two parasites. P. knowle-si has been described as the causal agent of life-threatening and lethal forms of malaria: its clini-cal picture is more severe when compared withthat of P. malariae, since the disease is character-ized by greater parasitaemia, as opposed to thatdocumented in the course of P. malariae disease.The most effective carrier is Anopheles leucos-phyrus: this mosquito is attracted by both humansand monkeys. Among primates, the natural hostsof P. knowlesi are Macaca fascicularis and Macacanemestina, while Saimiri scirea andMacaca mulatta,which cannot become infected in nature, may beuseful in experimental models. When underliningthe potentially severe evolution, we note the keyrole played by prompt disease recognition, which

is expected to be more straightforward in patientsmonitored in endemic countries at high risk, butshould be carefully implemented for subjects be-ing admitted to hospital in Western countries suf-fering from the typical signs and symptoms ofmalaria, after travelling in South-East Asia wherethey were engaged in excursions in the tropicalforest (trekking, and similar outdoor activities).In these cases, the diagnosis should be prompt,and suitable treatment should follow. Accordingto data in the literature, in non-severe caseschloroquine proves very effective against P.knowlesi, achieving the disappearance of signsand symptoms in 96% of cases after only 24 hoursafter treatment start. In the light of the emergingepidemiological data, P. knowlesi should be addedto Plasmodium vivax, Plasmodium ovale, P. malariae,and Plasmodium falciparum, as the fifth aetiologi-cal agent of malaria. During the next few years, itwill become mandatory to plan an appropriatesurveillance program of the epidemiological evo-lution, paying also great attention to the clinicalfeatures of patients affected by P. knowlesi malar-ia, which are expected to worsen according to thetime elapsed; some studies seem to point outgreater severity according to increased para-sitaemia, paralleling the increased interhuman in-fectious passages of the plasmodium.

SUMMARY

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Foci epidemici di malaria da Plasmodium knowlesi so-no stati individuati negli ultimi dieci anni in Malesia,in particolari negli stati di Sarawak e di Sabah (BorneoMalese) e nella regione di Pahang (Malesia peninsula-re). Sulla scorta della disamina della letteratura recen-te gli Autori sottolineano l’importanza dell’esame dia-gnostico di biologia molecolare (PCR, polymerasechain reaction), eseguito con primer specifici per P.knowlesi, in quanto si è osservato che all’esame micro-scopico tale plasmodio non ha caratteristiche peculiaried è difficilmente distinguibile dal Plasmodium mala-riae. Si è osservato che in alcuni casi il P. knowlesi puòportare al decesso, presentando un quadro clinico piùgrave di quello osservato nelle infezioni da P. malariaee presenta, come caratteristica laboratoristica differen-ziale, la tendenza a determinare parassitemie più ele-vate rispetto a quelle documentate nella malaria da P.malariae. Il vettore più efficace è l’Anopheles leuco-sphyrus; questa zanzara viene attirata sia dall’uomoche dalla scimmia. I primati ospiti naturali sono laMacaca fascicularis e la Macaca nemestina, mentre laSaimiri scirea e la Macata mulatta, che non si infetta-no in natura, possono essere utili come modelli speri-

mentali. Vista la possibile evoluzione clinica grave, sisottolinea l’importanza del riconoscimento precocenon solo nei pazienti ricoverati nelle zone a rischio maanche nei pazienti che si presentano negli ospedali oc-cidentali, dopo viaggi nel Sud-Est asiatico ove hannocompiuto escursioni nella foresta tropicale (trekking),accusando i sintomi tipici della malaria. In questi casila diagnosi deve essere precoce e si deve subito trattareadeguatamente il paziente. Secondo dati della lettera-tura, nei casi non gravi, la clorochina ha dimostratoessere farmaco molto efficace, determinando la scom-parsa dei sintomi dopo 24 ore dall’inizio del tratta-mento nel 96% dei casi. Alla luce dell’emergente qua-dro epidemiologico, il P. knowlesi si è aggiunto ai pla-smodi vivax, ovale, malariae, e falciparum, come quin-to agente etiologico dalla malaria. Nei prossimi annisarà necessario sorvegliare adeguatamente l’evoluzio-ne epidemiologica, valutando anche se le caratteristi-che cliniche dei pazienti con infezione da P. knowlesievolvono con il tempo verso un peggioramento; alcunistudi sembrano indicare una maggiore gravità, con au-mento dell’indice di parassitemia, aumentando il nu-mero dei passaggi infettivi interumani.

RIASSUNTO

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