Introduction

Over 120 Plasmodium species generally causing host-specific infections in mammals, birds and reptiles have been described [1]. The recognition of Plasmodium knowlesi as a protozoan parasite causing relevant malaria morbidity and mortality in humans has added two new aspects to our knowledge on malaria: a fifth species was added to the list of human malaria parasites and it has been established that human malaria is not necessarily caused by exclusively human-specific parasites.

P. knowlesi has first been studied in detail as a malaria parasite in monkeys in India in the early 1930s by Robert Knowles, Biraj Mohan Das Gupta and others. While long-tailed and pig-tailed macaques were found to be the natural hosts, it was soon observed that the parasite was also able to cause clinical disease in humans under experimental conditions [2, 3]. The first published natural infection in a traveller occurred in a US army member in 1965 after having spent 4 weeks in Peninsular Malaysia [4]. Yet, zoonotic malaria was considered as very rare until about 10 years ago. In 2004, a relevant proportion of clinical malaria had been attributed to P. knowlesi in Malaysian Borneo by recognising discrepancies between microscopic and molecular-genetic findings in human malaria cases [5]. Since then, P. knowlesi has been isolated from malaria patients in nearly all Southeast Asian countries including Malaysia, Thailand, the Philippines, Myanmar, Singapore, Vietnam, Indonesia, Brunei and Cambodia as illustrated in Fig. 1 [6–9••]. In some areas of Malaysian Borneo, P. knowlesi accounts for the majority of hospitalised malaria cases and the species has been shown to cause a high proportion of severe disease [10••].

Fig. 1
figure 1

Areas with reported Plasmodium knowlesi infections in humans and macaques as well as natural distribution of macaques being the natural host as well as of the responsible vector belonging to the Anopheles leucosphyrus group: Areas overlap [9••]. Numbers in parentheses indicate reported P. knowlesi cases in travellers to respective countries. Comment: instead of summarising all P. knowlesi cases recorded in that area, I suggest to provide numbers of P. knowlesi infections reported in international travellers, which would be as follows: Malaysian Borneo = 5, Peninsular Malaysia = 3, Thailand = 3, Philippines = 2, Indonesia = 1, Brunei = 1

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As P. knowlesi-endemic countries attract a major proportion of international travel, this review provides an overview on this Plasmodium species and summarises clinical evidence focussing on international travellers.

Parasite and Epidemiology

Of the five human Plasmodium species, P. knowlesi replicates most quickly with a life cycle of about 24 h. The parasite is phylogenetically closely related to Plasmodium vivax [11]. Both P. knowlesi and P. vivax use the Duffy red blood cell antigen as a receptor for cell invasion [12]. The rapid replication cycle leads to a quotidian fever after average prepatent and incubation times of 7, 2 and 11 days, respectively [1, 13]. Most human cases initially present with low parasite levels but the short replication time may rapidly lead to hyperparasitaemia [14]. Spontaneous remissions without antimalarial therapy have been described [13, 15]. As P. knowlesi, in contrast to phylogenetically related P. vivax, does not produce hypnozoites, relapses do not occur but re-infections with a different strain are possible [16, 17].

After first recognising P. knowlesi as a zoonotic malaria parasite contributing to a significant proportion of human malaria in 2004, P. knowlesi infections have been observed in every country in Southeast Asia except in Laos. P. knowlesi malaria appears to be more frequent in adults than in children as well as in males than in females which may reflect specific risk behaviour [9••]. Precise data on incidence and prevalence in Southeast Asia are lacking as it is morphologically difficult to differentiate P. knowlesi from Plasmodium malariae and Plasmodium falciparum by microscopy. In some areas of Malaysian Borneo where molecular genetic typing methods have been applied, all locally acquired malaria cases were attributable to P. knowlesi [9••]. In a recent study assessing parasites species in 453 samples positive for malaria parasites in 22 hospitals all over Malaysia, P. knowlesi was identified in 56 % of the samples followed by P. vivax (29 %), P. falciparum (11 %), Plasmodium ovale (<1 %) and P. malariae (<1 %) [18].

The natural hosts of P. knowlesi are long-tailed (Macaca fascicularis) and pig-tailed (Macaca nemestrina) macaques which are widely distributed throughout Southeast Asia. In some areas like Malaysian Borneo, up to 87 % of wild macaques have been shown to be infected with P. knowlesi [9••]. The main transmission cycle is largely confined to monkey-to-monkey transmission by forest-dwelling zoophilic anopheline mosquitoes of the Anopheles leucosphyrus group [9••]. In Malaysian Borneo, the area with the most intense P. knowlesi transmission, several Anopheles species competent for monkey-to-human transmission have been identified which predominantly feed between 7 and 10 p.m. [19–21]. Interestingly, P. knowlesi alone but also together with P. vivax and/or P. falciparum has been isolated from saliva glands of Anopheles dirus in southern Vietnam [22]. As A. dirus is the only known vector for human malaria transmission in this part of Vietnam, this observation indicates the potential for human-to-human transmission at least in some P. knowlesi-endemic areas [22].

As the habitats of the natural host and, correspondingly, of the Anopheles leucosphyrus group responsible for P. knowlesi transmission are located in forested areas, the main risk for human infections is confined to those entering forests for professional (e.g. farming, hunting, woodwork) or leisure (e.g. camping) activities [5, 14, 22]. As international travellers also enter these areas, an increasing number of P. knowlesi malaria cases have been published in the literature since 2005 (see Tables 1 and 2).

Table 1 Overview on epidemiologic characteristics of cases of imported Plasmodium knowlesi malaria in intercontinental travellers
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Table 2 Overview on clinical signs, course and outcome of imported Plasmodium knowlesi malaria in international travellers
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Diagnostics

Under the microscope, early trophozoites from P. knowlesi and P. falciparum are indistinguishable. Both parasite species can reach very high parasite loads; multiple infected red blood cells are characteristic. The more mature forms of P. knowlesi blood stage parasites including mature trophozoites and schizonts as well as gametocytes are similar to those of P. malariae. In fact, most P. knowlesi infections have been erroneously identified as P. malariae infections in routine microscopy [23–25]. Hence, blood microscopy is suitable for diagnosing plasmodial infection in general but it is of limited value in species differentiation in P. knowlesi-endemic areas. In contrast to P. malariae infection, P. knowlesi malaria can be life-threatening. Therefore, WHO recommends reporting P. malariae-positive blood films as P. malariae/P. knowlesi in areas endemic to the latter species [26].

Because currently used rapid diagnostic tests (RDTs) have been developed before the detection of P. knowlesi as a relevant cause of human malaria in Southeast Asia, their diagnostic specificity and sensitivity regarding this zoonotic Plasmodium species has not systematically been assessed. Results from tourists are inconclusive: RDTs containing pan-Plasmodium antibodies may react positive but sensitivity is lowest in P. knowlesi infections compared to other Plasmodium species. Pan-Plasmodium lactate dehydrogenase (pLDH) seems to have a slightly higher sensitivity as pan-Plasmodium aldolase (pALD) [27, 28•]. On the other hand, species-specific monoclonal antibodies for example against histidine-rich protein 2 (pfHRP-2) or lactate dehydrogenase of P. falciparum (pfLDH) or P. vivax (pvLDH) do not reliably react [9••, 28•]. In areas where P. vivax, P. falciparum and P. knowlesi co-exist, RDTs containing pfLDH or pvLDH may react positive due to the fact that P. vivax- and P. falciparum-LDH share 97 and >90 % homology with P. knowlesi-LDH [29]. In a recent study, the effectiveness of three commonly used RDTs was assessed in patients with P. knowlesi infection [30]. Using fresh blood samples from patients with microscopically or PCR-confirmed infections, OptiMAL-IT showed the highest sensitivity of 71 % (95 % confidence interval (CI) 54–88 %—yielding predominantly P. falciparum-positive results) compared to BinaxNOW® Malaria and Paramax-3 RDT with a sensitivity of 29 % (95 % CI 12–46 %) and 40 % (95 % CI 21–59 %), respectively. Even the combination of two RDTs containing pLDH/pfLDH (OptiMAL-IT) and pLDH/pfHRP (CareStart) did not increase the sensitivity which was 25 % (95 % CI 19–32 %) but increased the specificity to 97 % (95 % CI 92.00 %) [31]. The sensitivity seems to be higher in severe malaria cases possibly due to higher parasite levels. To date, no P. knowlesi-specific antigen suitable for RDTs in endemic areas has been identified [25]. All in all, the additive value of RDTs in diagnosing P. knowlesi infections remains uncertain—in particular, in very low parasite levels.

A series of PCR-based molecular-genetic detection methods has been established but these are usually not broadly available for routine diagnostics in particular in resource-poor settings. Even in Western countries, PCR-based malaria diagnostics are limited to specialised parasitology laboratories. Nested PCR assays are widely used to ascertain Plasmodium species but this approach is labour-intense, involving a PCR each for all five parasite species. Furthermore, previously used nested PCR assay developed for the detection of P. knowlesi have been modified due to their false-positivity in P. vivax infections indicating cross-reactivity and limited species-specificity of previously used molecular-genetic methods [32, 33]. Very sensitive real-time multiplex PCR assays offer a more rapid diagnostic approach but also require more sophisticated laboratory equipment. A single-step hexaplex PCR system targeting the 18S ssu-rRNA of all five human Plasmodium species that overcomes some of the obstacles of the previous two methods has recently been described [34].

Due to these diagnostic difficulties related to microscopy and RDTs as well as the limited availability of molecular-genetic methods beyond research laboratories in endemic areas it can be anticipated that the current knowledge on P. knowlesi epidemiology is still incomplete. The lack of specific detection methods in P. knowlesi-endemic tourist destinations and in community hospital settings in the traveller’s home country should not delay rapid initiation of antiparasitic treatment given the fact that P. knowlesi replicates quickly and infection may progress to life-threatening disease.

Clinical Disease

Clinical signs and symptoms of acute P. knowlesi malaria are non-specific and frequently include daily fever and chills corresponding with the short erythrocytic parasite cycle. Accompanying symptoms may include headache, malaise, reduced appetite, abdominal pain, diarrhoea and even cough. While most patients presented to a health care facility within 4–5 days, some patients were ill for several weeks before seeking medical care [10••, 35–37]. Upon clinical examination, the most common findings include tachypnea and tachycardia in association with fever. In a quarter to a third of patients, enlarged liver and spleen have been reported [10••, 35].

Nearly all patients are thrombocytopaenic upon or shortly after hospitalisation. Nevertheless, bleeding complications are rare [35]. Pronounced thrombocytopaenia may occur at low parasite levels in P. knowlesi malaria. Therefore, careful examination of blood films is necessary before excluding P. knowlesi malaria in these patients [9••]. While anaemia occurs, severe anaemia is less frequent compared to P. falciparum malaria [35]. Renal impairment has been described in 6.9 and 14.5 % in two recent prospective studies on 130 and 110 patients with P. knowlesi malaria but is usually reversible [10••, 38••].

P. knowlesi infection can cause complicated and even life-threatening malaria. In fact, recent prospective case-control studies conducted in Malaysian Borneo indicate that up to 29 % of patients with P. knowlesi malaria progress to severe disease rendering the proportion of patients with severe malaria (applying severe P. falciparum malaria definitions) threefold higher compared to patients with P. falciparum malaria in endemic areas [10••, 38••]. Complications include parasite levels >100.000/μl, jaundice, acute respiratory distress, hypotension, acute renal failure, acidosis and, rarely, hypoglycaemia [10••, 35]. A parasitaemia of ≥35.000/μl or 1 % infected red blood cells and a thrombocytopaenia of ≤45.000/μl have been associated with a ten- and fivefold higher risk of developing complications, respectively, in adult patients with P. knowlesi malaria [38••]. Cerebral malaria, the complication associated with highest mortality in severe P. falciparum malaria, has not been described in P. knowlesi malaria. High parasite loads have been shown to be associated with complicated disease but severe disease can also occur at lower parasite levels [10••, 38••].

Treatment

No randomised clinical trial has been conducted to assess the efficacy of antimalarials in P. knowlesi malaria. Accordingly, none of the antimalarials has been licensed for its treatment in the Western world. Currently, available evidence, mainly obtained from case series to smaller prospective observational studies, covers a wide range of antimalarial drugs and indicates that most antimalarials are effective [9••].

Cases with uncomplicated P. knowlesi malaria have been successfully treated with virtually all conventional antimalarials including chloroquine, mefloquine, atovaquone-proguanil, artemether-lumefantrine, quinine and intravenous artesunate [9••], see also Table 2. A randomised study comparing artesunate-mefloquine combined therapy versus chloroquine in uncomplicated P. knowlesi malaria is expected to complete recruitment at the end of 2014 but it is unlikely that mefloquine will play a relevant role in treating P. knowlesi due to better-tolerated alternative drugs [39]. P. knowlesi is considered as sensitive to chloroquine [40•]. From a practical approach, however, this antimalarial does not seem to be a suitable prophylactic or therapeutic option as P. knowlesi-endemic areas have the highest proportion of antimalarial drug-resistant strains of other malaria parasites, in particular, P. falciparum, while rapid and reliable species-differentiation can not be warranted outside specialised centres. In addition, ex-vivo assessment of the sensitivity of human P. knowlesi isolates indicate high sensitivity to artemisinins, variable and moderate sensitivity to chloroquine, and lower sensitivity to mefloquine [41•].

The WHO favours a pragmatic approach by recommending artemisinin-based combination therapies (ACT) generalising existing evidence as well as data derived in particular from P. falciparum malaria to P. knowlesi infections [26]. German guidelines recommend an ACT or atovaquone-proguanil following the therapeutic approach for P. falciparum malaria [42]. The US Centers of Disease Control and Prevention (CDC) currently recommend chloroquine for treating P. knowlesi malaria [43]. As P. knowlesi infection may rapidly lead to high parasitaemia, it seems reasonable to treat promptly with a fast-acting antimalarial. Despite the fact that oral ACTs available for treatment of uncomplicated malaria in the Western world (including artemether-lumefantrine (Riamet®, Coartem®) or dihydroartemisinin-piperaquine (Eurartesim®)) are not licensed for P. knowlesi infection, an oral ACT seems to be the drug of choice in cases in which P. knowlesi malaria has to be considered in particular when the species identification can not be rapidly and reliably secured in specialised centres. This practical approach is supported by a recent publication from Malaysian Borneo showing that of six deaths related to P. knowlesi malaria, all had initially been reported as P. malariae infection by microscopy and only two received immediate parenteral antimalarial treatment [44••].

For severe P. knowlesi malaria, retrospectively collected data indicate a superior effect on parasite clearance time as well as survival of intravenous artesunate versus quinine which is in line with existing evidence derived from large trials on severe P. falciparum malaria [45]. A recently published prospective study conducted in Malaysian Borneo indicates that early referral and prompt initiation of intravenous artesunate is highly effective also in severe P. knowlesi malaria [10••]. Whether complications like delayed haemolysis after artesunate therapy, in particular in patients with high parasite loads, also occur in P. knowlesi malaria is unclear but it seems to be justified to follow-up on haemoglobin levels at 2 and 4 weeks after initiation of intravenous artesunate in particular in those patients treated with high parasite levels (e.g. >5 % infected red blood cells) [46].

P. knowlesi has so far mainly been considered a zoonotic disease and its relevance in human malaria has only recently been established. Yet, this does not exclude existing drug resistance per se as human cases may have broadly been mistaken as P. malariae (or P. falciparum) infections in the past and may have been exposed to respective antimalarials in a relevant dimension. In fact, parasite levels continued to increase despite mefloquine therapy in a recent case report which supports the observation, that antimalarial resistance can easily be induced by repeated drug exposure to P. knowlesi infected rhesus macaques [17, 47, 48].

P. knowlesi should be considered in all travellers returning with microscopically diagnosed P. malariae (or P. falciparum) infection and parasite levels should be assessed. However, antimalarial treatment should not be delayed until the causative species has been ascertained as P. knowlesi may progress to high parasitaemia quickly resulting in complicated, life-threatening disease. Despite lacking respective evidence, an ACT like artemether-lumefantrine or, alternatively, atovaquone-proguanil should be the treatment of choice. Transmission areas for P. knowlesi and dengue virus overlap with dengue fever being the suspected primary diagnosis in many febrile patients with thrombocytopaenia. Therefore, dengue virus infection should be considered and ruled out.

Role in Travellers/Prophylaxis

The first case of natural P. knowlesi infection was described in a US army surveyor in 1965 after returning from a 4-week trip to Peninsular Malaysia [4]. Since then and after P. knowlesi has been recognised to contribute to human malaria in Southeast Asia, 12 cases have been reported in intercontinental travellers between 2006 and 2013, see Tables 1 and 2. The UK Health Protection Agency mentions one additional case of P. knowlesi malaria reported in a traveller to Brunei without providing any further information [40•]. Two cases have been reported in international travellers within Asia: a Taiwanese traveller to Palawan Island, the Philippines, in 2005 and a Japanese traveller to Malaysia in 2012 [49, 50]. Except for the one case reported from Brunei, countries of infection were Malaysia (n = 8 with three cases from Peninsular Malaysia and five cases from Malaysian Borneo), Thailand (n = 3), the Philippines (n = 2) and Indonesia (n = 1). One case had travelled to several countries including Thailand, Indonesia, Malaysia and Vietnam. For epidemiologic and clinical details on all 15 informative cases in international travellers see Tables 1 and 2, respectively.

Male gender and trips to forested areas inhabited by monkeys have been described as risk factors for infection [9••]. This is in line with the characteristics of the 15 P. knowlesi infections reported in international travellers so far (Table 1). Eleven (73 %) travellers were male, median age was 40.4 years (range 32–60). Almost all travellers had visited rural/forested areas; several reported that they had seen wild monkeys during their journey. The travel duration ranged from 10 days to 18 months indicating that P. knowlesi malaria also occurs in short-term travellers. None of the travellers had taken regular/continuous malaria prophylaxis. As shown in Table 2, 85 % were thrombocytopaenic upon hospitalisation. While one patient became symptomatic 3 days before leaving the endemic country, the mean time from last potential exposure (leaving the endemic country) to disease onset was 7.4 days (range 0–13). The initial treatment in the 15 travellers with P. knowlesi malaria was atovaquone-proguanil (n = 5), chloroquine (n = 5), mefloquine (n = 2), intravenous quinine (n = 1) and intravenous artesunate (n = 1). One patient who took irregular malaria prophylaxis recovered apparently without antimalarial therapy. One case was complicated by jaundice; in another patient, hypoglycaemia occurred most likely related to the quinine treatment and the other 13 cases were uncomplicated—all survived.

Travellers to P. knowlesi-endemic areas, in particular, those intending to travel to rural, forested areas should be informed about the risk as well as respective exposure prophylaxis measures including repellents, bed nets etc. Current commercially available RDTs are unreliable in detecting P. knowlesi infection. While epidemiologic data on risks in local populations is still incomplete, specific risks in travellers are unknown. In the author’s opinion, continuous malaria prophylaxis is not generally warranted in travellers to P. knowlesi-endemic areas but in particular, travellers at higher risk of infection (outdoor activities, ecotourism, stay in rural/forested areas, anticipated contact with wild monkeys) should be provided with advice to seek medical advice as soon as possible after onset of fever during or after travel and should be provided with either an ACT or atovaquone-proguanil as antimalarial drug for stand-by emergency treatment. Recommendations for malaria prevention in travellers to P. knowlesi-endemic areas according to German, US and UK guidelines are summarised in Table 3.

Table 3 Recommendations for malaria prevention in selected Plasmodium knowlesi-endemic areas with reported cases in travellers
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Conclusion

P. knowlesi occurs in all Southeast Asian countries except Laos. Microscopy is useful to detect plasmodial infections but is of limited value for species differentiation. Rapid diagnostic tests are not reliable in detecting P. knowlesi infection because of low sensitivity. In patients returning with malaria from Southeast Asia—in particular in those with P. falciparum and P. malariae-positive blood film readings—molecular-genetic typing should be requested. Yet, rapid initiation of antimalarial treatment should not be delayed. Patients with P. knowlesi malaria may proceed to high parasite levels rapidly with the potential for life-threatening disease. Despite lacking respective evidence, an ACT seems to be the drug of choice for treating P. knowlesi infections. Travellers to endemic countries—in particular those intending to visit forested/rural areas or anticipate contact with wild monkeys—should be advised about risks and preventive measures including the fact that reliable diagnosis should sought rapidly while RDTs are unreliable.