Human Infections with Plasmodium Knowlesi-Zoonotic Malaria

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Human infections with Plasmodium knowlesi—zoonotic malaria

S. B. Millar and J. Cox-Singh School of Medicine, University of St Andrews, Medical and Biological Sciences Building, North Haugh, St Andrews, Fife, UK

Abstract

In 2004 a large focus of Plasmodium knowlesi malaria was reported in the human population in Sarawak, Malaysian Borneo. Plasmodium knowlesi, a parasite of the South-East Asian macaques (Macaca fascicularis and Macaca nemestrina), had entered the human population. Plasmodium knowlesi is transmitted by the leucosphyrus group of Anopheline mosquitoes and transmission is largely zoonotic and restricted to the jungle setting. Humans entering jungle transmission sites are at risk. Since 2004, human cases of P. knowlesi have been continuously reported in local communities and in travellers returning from South East Asia. Plasmodium knowlesi is the most common type of indigenous malaria reported in Malaysia. Infections are most often uncomplicated but at least 10% of patients report with severe malaria and 1–2% of cases have a fatal outcome. Parasitaemia is positively associated with the clinical and laboratory markers of severe malaria. The current literature on P. knowlesi, including epidemiology, natural hosts and vectors, pathogenesis, clinical descriptions, treatment and diagnosis, is reviewed. There are many gaps in our understanding of this disease that are highlighted here with suggestions for further research to inform pre-emptive control measures that would be required to prevent a full emergence of this parasite into the human population. Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Keywords: Diagnosis, epidemiology, malaria, pathogenesis, Plasmodium knowlesi, transmission, treatment, vectors, zoonotic Article published online: 2 April 2015

adapted to one vertebrate host species tend not to cross into Corresponding author: J. Cox-Singh, School of Medicine, Univer- other hosts even when two hosts have very similar parasites sity of St Andrews, Medical and Biological Sciences Building, North Haugh, St Andrews, Fife, KY16 9TF, UK [3]. An exception to this is Plasmodium knowlesi, a parasite E-mail: [email protected] adapted to the South-East Asian macaques. Plasmodium knowlesi was discovered in 1932 in Macaca fascicularis (long-tailed macaque) and since then has been maintained experimentally in Introduction Macaca mulata (Rhesus macaques) and was successfully trans- ferred to other non-human primates and human volunteers under experimental conditions [4,5]. Naturally acquired Malaria is caused by parasites that are member species within P. knowlesi infections in the human population were thought to the genus Plasmodium. Plasmodium falciparum, Plasmodium vivax, be rare with only one conﬁrmed case, in 1965 [6]. Plasmodium ovale (exists as two subspecies) and Plasmodium Plasmodium knowlesi is morphologically similar to the human- malariae account for almost all cases of malaria in the human host-adapted P. malariae that is transmitted between human population [1]. They are human-host adapted and are trans- hosts and causes mild and often self-resolving malaria with low mitted from person to person by female Anopheline mosqui- parasite counts (the number of parasite-infected red blood toes and, as is typical of the genus, grow and replicate inside the cells/μL of blood) [4]. A large entry of P. knowlesi was discov- vertebrate host red blood cells. Plasmodium species are not ered in the human population in the Kapit district of Sarawak, restricted to human infections and more than 100 species have Malaysian Borneo during an investigation of atypical cases of evolved and co-exist with other vertebrates including birds, P. malariae, where patients were reporting with symptoms and rodents and non-human primates [2]. Plasmodium species uncharacteristically high parasite counts (parasitaemia) [7].

Clin Microbiol Infect 2015; 21: 640–648 Clinical Microbiology and Infection © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rightsreserved http://dx.doi.org/10.1016/j.cmi.2015.03.017 CMI Millar and Cox-Singh Plasmodium knowlesi malaria 641

Parasite DNA from malaria patients with atypical P. malariae Studies so far tend to confirm that P. knowlesi transmission is infections was sequenced [7]. The sequences aligned with largely restricted to the jungles of South-East Asia because this P. knowlesi reference sequences and were quite distinct from is the preferred habitat of the particular Anopheline vectors of P. malariae. Patients with all-cause malaria were then recruited P. knowlesi [34–36]. People who are engaged in agriculture, and their malaria parasites were identified using Plasmodium forestry, hunting or leisure activities in the jungle setting are at species-specific PCR. Of the 208 patients in the study, risk. P. knowlesi was the most common cause of malaria [7].Since then there have been studies on the prevalence and distribu- The natural hosts of P. knowlesi tion of P. knowlesi in South-East Asia and even though P. malariae is still reported by microscopy, molecular methods suggest that P. malariae is rare in South-East Asia and P. knowesi Parasitologists in the twentieth century found that the pre- malaria is widespread [8–10]. Since the report of the discov- dominant simian hosts of P. knowlesi are M. fascicularis (long- ery of P. knowlesi malariainthehumanpopulationin2004the tailed macaques) and Macaca nemestrina (pig-tailed macaques), disease has been clinically characterized and P. knowlesi is now which are indigenous to South-East Asia [4,37]. However, recognized as the fifth cause of malaria in humans [11].Here although these macaques are suspected of being the natural we review the current literature on P. knowlesi including reservoirs for the human P. knowlesi infections reported over epidemiology, natural hosts and vectors, pathogenesis and the past 10 years, parasitology data from local macaque pop- diagnosis. ulations are sparse. Nonetheless P. knowlesi has been detected in wild populations of M. fascicularis in Malaysia, Singapore and Thailand, and M. nemestrina in Malaysia and Thailand Epidemiology [17,18,38–40]. For example, in Peninsular Malaysia 143 M. fascicularis individuals were trapped and screened for Following the discovery of P. knowlesi in the Kapit district of P. knowlesi by PCR [39]. Ten animals were positive for Sarawak, blood samples from patients with malaria at other P. knowlesi [39]. In Singapore, three M. fascicularis monkeys, sites in Malaysia were screened using Plasmodium species- trapped in an area where humans were thought to have been specific PCR. Surprisingly the entry was not restricted to the infected, had multiple P. knowlesi genotype infections whereas Kapit district but cases of P. knowlesi malaria were widespread ten macaques from a nature reserve were all negative for in Sarawak, Sabah, also Malaysian Borneo, and mainland P. knowlesi [17]. A recent molecular study on the macaques in Malaysia and P. malariae was rare [8]. The Plasmodium species the Kapit division of Sarawak, Malaysian Borneo, where the first normally associated with malaria in humans, P. falciparum and large focus of P. knowlesi was identified in the human population P. vivax, are largely under control in island and mainland [7], found that M. fascicularis and M. nemestrina macaques were Malaysia and currently P. knowlesi is the main cause of indige- infected with multiple Plasmodium species including P. knowlesi, nous malaria in Malaysia [10]. Plasmodium inui, Plasmodium cynomolgi, Plasmodium fieldi and The number of cases of P. knowlesi malaria in Sarawak is not Plasmodium coatneyi [38]. Additionally, some of the macaques in decline and the number of notified cases of P. knowlesi in were infected with multiple genotypes of P. knowlesi [38]. Plas- Sabah increased from 703 in 2011 to 996 in 2013 [9]. Addi- modium knowlesi DNA sequences from macaques and human tionally, P. knowlesi appears to have increased in the Hulu cases of P. knowlesi in the same area were compared. There was Selangor district of Peninsular Malaysia from 21 cases in 2009 to no evidence of clustering of P. knowlesi genotypes in either host 41 cases in 2013, peaking at 43 cases in 2011 [12]. species that would indicate a host switch event of the parasite In addition to Malaysia, P. knowlesi has been identified across from macaques to humans [38]. The fact that parasite geno- South-East Asia with cases confirmed in Cambodia, Indonesia, types seemed to be shared between macaques and humans Myanmar, Philippines, Singapore, Thailand and Vietnam supports the idea that, in the Kapit division of Sarawak, [13–20]. Additionally, P. knowlesi cases were reported in P. knowlesi remains zoonotic [38]. The South-East Asian ma- Australia, Finland, France, Germany, Japan, the Netherlands, caque populations are relatively large and the main reservoir of New Zealand, Scotland, Spain, Sweden and Taiwan in travellers P. knowlesi, but other non-human primates may well be infected. returning home from P. knowlesi transmission zones in South- In Thailand, blood collected from a leaf monkey, Semnopithecus East Asia [14,21–33]. Taken together with the figures from obscurus, was positive for Plasmodium species with similarity to Malaysia and the wide distribution of cases reported across P. knowlesi [41]. In earlier studies the banded leaf monkey was South-East Asia, the multiple entries of P. knowlesi into the identified as one of the natural hosts of P. knowlesi [18,41,42]. human population appears to be sustained. The macaque populations in South-East Asia are currently

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successful and multiple independent entries of P. knowlesi into A. dirus mosquitoes were multiply infected with P. knowlesi, the human population are observed across South-East Asia. P. falciparum, P. vivax and P. malariae [20]. This information, taken together with the potential for severe The complexity and diversity of the leucosphyrus group of disease and the uncertainty of a host-switch event having taken mosquitoes in South-East Asia precludes extrapolation of in- place, it would seem pertinent to generate more data on the formation between locations. Therefore collecting represen- reservoirs of P. knowlesi that would be representative of the tative information on the local vectors of P. knowlesi within the region. Recently a model was developed to predict the likeli- region is important to identify areas where the human popu- hood of P. knowlesi malaria occurring in specific areas within lation is at risk [40], and also to monitor changes in vector South-East Asia [40]. However, local information on reservoirs behaviour that may predict a host switch for P. knowlesi from of infection, vectors and human cases of P. knowlesi is required macaques to human-to-human transmission. to enter into the model [40]. Generating this kind of data, particularly on the vectors and reservoirs of P. knowlesi is not Plasmodium knowlesi pathogenesis and trivial, but if the goal is to predict and monitor unusual outbreaks of P. knowlesi in South-East Asia then representative data parasite diversity are essential. In malaria it is unclear why certain individuals develop severe Anopheline vectors of P. knowlesi disease while others present with uncomplicated infections. Studies can be equivocal but it is certain that both host and parasite variability contribute to severe malaria [48]. For Transmission of P. knowlesi is restricted to the Anopheles leu- example, P. falciparum and P. knowlesi reach very high parasite cosphyrus group of mosquito vectors that are found in South- counts in some patients and hyperparasitaemia is associated East Asia [4,43,44]. The group is diverse with many species with severe disease in both of these types of malaria [49]. found across the region, each with differing vectorial capacities However, it is not known if some hosts are particularly sus- [43]. The group includes vectors not only of P. knowlesi but ceptible to Plasmodium infection or if some parasites grow more also of P. falciparum and P. vivax. Although the number of efficiently or are more virulent in the human host. studies is small, mosquitoes at suspected jungle transmission sites of P. knowlesi, including those in Malaysian Borneo Hyperparasitaemia and invasion efficiency [35,45–47], Hulu Selangor (Peninsular Malaysia) [12],Kuala Patients with P. knowlesi malaria present with a range of para- Lipis (Peninsular Malaysia) [36,39] and Khanh Phu (South sitaemias from 1 to 764 720/μL, even though the duration of Vietnam) [20] were sampled for P. knowlesi carriage. Anopheles symptoms before presentation can be relatively short [8].A balabacensis is implicated in P. knowlesi transmission in Sabah recent study examined the possibility that variation in Malaysian Borneo [46,47]. In Sarawak the main vector of P. knowlesi proteins involved in parasite invasion of host P. knowlesi in the Kapit Division was Anopheles latens [35]. erythrocytes may confer an invasion advantage to particular Anopheles latens feeds on humans and long-tailed macaques and parasite genotypes and lead to high parasite counts [50]. Poly- was therefore incriminated as the vector of P. knowlesi in this morphic regions of the two P. knowlesi members of an impor- region of Sarawak [35]. In Hulu Selangor, peninsular Malaysia, tant invasion gene family in Plasmodium species (P. knowlesi Anopheles maculatus was the predominant malaria vector normocyte binding protein (Pknbp) xa and Pknbpxb) [51] were species and one of 265 mosquito samples had P. knowlesi oo- sequenced in patient isolates [50]. Particular alleles of both cysts [12]. Two studies from Kuala Lipis, 100 km from Hulu genes clustered with clinical and laboratory markers of severe Selangor, reported that Anopheles cracens, also within the leu- disease [50]. Although this work is preliminary, the results cosphyrus group, was the predominant malaria vector [36].Five suggest that certain genetic variants of P. knowlesi are more A. cracens were positive for P. knowlesi sporozoites or oocysts, virulent in the human host. two of these were obtained by human bait collection [36,39]. One of these studies included a village setting where A. cracens, Parasite sequestration in P. knowlesi A. maculatus and Anopheles aconitus were all captured outdoors Severe malaria with coma in P. falciparum malaria is accom- but none of these species were found inside human dwellings, panied by sequestration of parasite-infected erythrocytes in therefore reducing the likelihood of human-to-human trans- the brain [52]. Although incompletely understood, seques- mission of P. knowlesi within households [36]. In Khanh Phu, tration in P. falciparum is mediated by the expression of the var southern Vietnam, Anopheles dirus was identified as the pre- gene family of proteins, PfEMP1, on the surface of infected dominant malaria vector species [20]. Interestingly some erythrocytes [53–56]. Plasmodium knowlesi parasites express

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variant schizont-infected cell agglutination variant proteins on difference in the proportion of patients with severe disease the surface of infected erythrocytes that are also encoded by a (9–39%) at the different study sites reflects recruitment at variant multigene family [55–60]. PfEMP1 proteins predomi- peripheral hospitals [34,69] and at a referral hospital where nantly bind to CD36, a receptor that is constitutively the proportion of severe cases would be expected to be expressed on human endothelial cells, except on brain higher [68,71]. In addition parasite diversity and associated endothelium where they bind to up-regulated intercellular virulence at different study locations may affect the proportion adhesion molecule 1 and to each other to form rosettes of patients with severe disease (see Hyperparasitaemia and [61–65]. Severe malaria with coma has not been reported in Invasion efficiency section) [50]. Parasitaemia is associated severe cases of P. knowlesi malaria even though sequestration with disease severity in P. knowlesi infections [34] and in three of parasitized erythrocytes, similar to that found in severe studies the geometric mean parasitaemia in uncomplicated falciparum malaria with coma, was observed in post mortem P. knowlesi malaria was between 1030 and 4837 parasites/μLof brain sections taken from a fatal case of P. knowlesi malaria blood [68–70]. However for severe disease the geometric [66]. A subsequent study found that P. knowlesi-infected mean parasitaemia was 94 256 (18 500–290 000 interquartile erythrocytes, matured ex vivo, bound to intercellular adhesion range) [69],80359(26000–170 000 interquartile range) [68] molecule 1 and/or vascular cell adhesion molecule 1 but not and 21 715 (4300–150 000 interquartile range) [70] parasites/ CD36 [67]. The study was small but highlighted the potential μL, respectively. Thrombocytopenia is a characteristic feature not only for sequestration but also cytoadherence in of P. knowlesi infection [34,72]. All studies that compared un- P. knowlesi malaria. Further work on schizont-infected cell complicated with complicated P. knowlesi malaria reported agglutination variants and their contribution to P. knowlesi even lower platelet counts in patients with severe disease virulence may well provide insight into the role of parasite [68–71]. The clinical descriptions of P. knowlesi were recently sequestration, cytoadherence and disease severity, including reviewed in detail (see Singh & Daneshvar [73]), and in severe disease with coma, in malaria. returning travellers (see Muller & Schlagenhauf [74]). Patients with severe knowlesi malaria fulfil the WHO criteria for severe malaria apart from severe anaemia and coma [49]. Clinical presentation Four fatal cases of P. knowlesi malaria were reported in 2008 [8]. Since then prospective and retrospective studies have recorded Plasmodium knowlesi infections are characterized by daily, mortality rates of 1.8–10.7%; however, the higher figures are symptomatic episodes because of the unique 24-h asexual biased by recruitment in a referral hospital [68–71]. Prospec- (erythrocytic) life cycle of P. knowlesi. The asexual life cycle tive studies at district hospital level suggest that 1–2% of pa- takes at least 48 h in all of the other Plasmodium species that tients with knowlesi malaria have a fatal outcome [34]. infect humans and non-human primates. The first detailed Importantly a recent study in Sabah reported no deaths in all clinical descriptions of P. knowlesi malaria were provided by patients with P. knowlesi who were ill enough to require referral Daneshvar et al. [34]. The study included baseline pre- to a specialist centre, when they received pre-referral intra- treatment signs, symptoms and clinical laboratory data for a venous artesunate [68]. Intravenous artesunate had a faster patient cohort (n = 107) in Kapit, Sarawak [34]. The predom- parasite clearance time when compared with treatment with inant symptoms were; fever and chills (100% of patients), intravenous quinine [71] and parasites responded uniformly headache (94.4%), rigors (89.7%), malaise (89.7%), myalgia well ex vivo to artesunate when compared with chloroquine, (87.9%), cough (56.1%), nausea (56.1%), abdominal pain mefloquine and artemisinin [75]. Interestingly in one of our (52.3%), vomiting (33.6%) and diarrhoea (29%) [34]. Patients recent studies a 57-year-old female patient presented with had a median axillary temperature of 37.6°C, median respira- 794 000 parasites/μL blood with numerous multiply infected tory rate of 26 breaths/min, mean pulse rate of 95 beats per erythrocytes. On admission her white cell count was 16 500/μL minute and mean arterial blood pressure of 89 mmHg [34]. Ten (83% neutrophils), haemoglobin 9.1 g/dL, packed cell volume of the 107 (9.3%) patients had severe malaria but none had 25%, platelets 39 000/μL, serum creatinine 359 μmol/L, serum severe malaria with coma [34]. urea 32 mmol/L, total serum bilirubin 56.1 μmol/L with Subsequently four studies compared patients presenting 32.4 μmol/L as conjugated bilirubin. The patient was treated with uncomplicated and severe knowlesi malaria to determine with intravenous artesunate in combination with doxycycline the clinical and laboratory markers of severe disease. The and survived, and although not enrolled in a study on treatment studies comprised 130 [68],110[69],94[70] and 56 [71] response, this case supports the important recommendation by patients, of whom 38 (29%), 17 (15%), 9 (9.5%) and 22 Barber et al., for the use of IV artesunate to treat severe (39%), respectively, had severe disease [68–71].The P. knowlesi malaria [68].

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P. knowlesi diagnosis and prompt diagnosis and treatment. Misdiagnosing P. knowlesi as P. malariae can have severe consequences [8,77]. In addition to the absence of morphological characteristics to distinguish Traditionally malaria is diagnosed by examining stained thick P. knowlesi from P. malariae, microscopy is error prone and and thin blood films by microscopy. Malaria parasites are visible other species can also be misdiagnosed [7,8,78,79]. The prob- as stained bodies inside red blood cells (Fig. 1). The human host lems encountered with misdiagnosis when using only micro- adapted parasites P. falciparum, P. vivax, P. malariae and P. ovale scopy highlight the need for the development of sensitive and have unique morphological characteristics that are used to specific methods to accurately diagnose malaria in a timely identify the species of Plasmodium responsible. However, the fashion. entry of P. knowlesi into the human population has complicated diagnosis based on parasite morphology. Plasmodium knowlesi Antigen-based rapid diagnostic tests for malaria and P. malariae share morphological characteristics and there- Antigen-based rapid diagnostic tests (aRDTs) for malaria were fore cannot be differentiated by microscopy. Plasmodium introduced to provide bedside diagnosis without the need for malariae replicates every 72 h, preferentially invades older microscopy. Several commercially available aRDTs include a erythrocytes, has a low parasitaemia and patients do not Pan-Plasmodium reagent to detect P. falciparum, P. vivax, develop severe symptoms [76], therefore misdiagnosis is not a P. malariae and P. ovale in addition to a P. falciparum-specific problem. However, P. knowlesi replicates every 24 h, can rapidly reagent (Table 1). The aRDTs were recently evaluated for reach high parasitaemias and can cause severe and fatal malaria cross-reactivity with P. knowlesi [80–83]. The OptiMAL-IT [8]. Therefore patients with P. knowlesi malaria require accurate assay had the highest sensitivity for fresh and frozen whole

FIG. 1. Red blood cells infected with asexual stage parasites in Giemsa-stained thin blood ﬁlms from patients with Plasmodium knowlesi malaria: (a) early trophozoite (ring stage); (b) trophozoite with increased blue-stained cytoplasm and diffuse hemozoin pigment grains (yellowish areas at the periphery of the cytoplasm); (c) schizont containing ten merozoites, daughter parasites, with clumped pigment present (densely stained body); and (d) schizont undergoing egress to release merozoites to infect new red blood cells. The complete erythrocytic cycle from merozoite invasion of a target red blood cell to schizont rupture in P. knowlesi takes 24 h.

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TABLE 1. Summary of the antibody reagents used in commercially available antigen detection rapid diagnostic tests (aRDTs) for malaria diagnosis

Commercially available aRDTs Pan Plasmodium reagent Plasmodium falciparum reagent Plasmodium vivax reagent Reference

OptiMAL-IT pLDH PfLDH — [80,81,83] (DiaMed) BinaxNOW® Malaria pAldolase PfHRP2 — [81] (Inverness Medical) Paramax-3 pLDH PfHRP2 PvLDH [81] (Zephyr Biomedical systems) CareStart™ Malaria pLDH PfHRP2 — [80] (Access Bio, Inc.) ParaHIT® pAldolase PfHRP2 — [82] (Span Diagnostics Ltd.) First Response® pLDH PfHRP2 — [82] (Premier Medical Corporation Ltd.) Entebe — PfHRP2 PvLDH [83] (Laboratorium Hepatika)

blood from PCR-confirmed single-species P. knowlesi infections, PCR for P. knowlesi detection 71% (20/28) and 73% (30/41), respectively [81]. However, 90% Currently, PCR is the only method that can accurately identify (18/20) of the Pan-positive P. knowlesi samples were also posi- P. knowlesi parasites. Nested-PCR was required to improve tive for P. falciparum PfLDH. Only 10% of the P. knowlesi sam- sensitivity, particularly when detecting P. knowlesi in isolates ples were identified as non-P. falciparum malaria using from patients with low parasitaemia [7]. Often nested-PCR is OptiMAL-IT [81]. Therefore the OptiMAL-IT test was not used to retrospectively confirm the Plasmodium species when recommended for diagnosing malaria in areas where P. knowlesi P. knowlesi is suspected [8,10,13,16,26,32,34–36]. However, malaria occurs [81]. The BinaxNOW® and Paramax-3 tests although nested-PCR is specific it is not practical for malaria both had low sensitivities for P. knowlesi and BinaxNOW® was diagnosis [79,84]. A sensitive multiplex quantitative PCR assay negative for all samples <5000 parasites/μL [81]. Additionally, for malaria, that includes P. knowlesi detection, has been P. knowlesi samples cross-reacted with the P. vivax LDH developed with 100% sensitivity and specificity for P. knowlesi (PvLDH) reagent in the Paramax-3 test kit [81]. Cross- [85]. This assay requires a five-colour real-time PCR cycler and, reactivity between P. knowlesi and the OptMAL-IT PfLDH re- although rapid, is practically prohibitive for malaria diagnosis in agent and P. knowlesi with the Entebe MC PvLDH reagent was endemic countries. In addition, processing one sample at a time previously reported when aRDTs were evaluated using for rapid diagnosis of individual patients presenting with fever P. knowlesi-infected Japanese macaques [83]. Plasmodium knowl- would also be impractical. However, for epidemiology and PCR esi did not react with PfHRP2 antibodies but reacted with confirmation this assay has the advantage of speed and is a PvLDH as in the Paramax-3 assay [81,83]. Further studies have single-tube assay. The commercially available PlasmoNex suggested that P. knowlesi identification can be achieved using a multiplex PCR system for malaria includes species-specific combination of OptiMAL-IT and CareStart™ aRDTs; however, primers for P. knowlesi but requires gel electrophoresis correct P. knowlesi identification was still less sensitive than [86,87]. Although specific and sensitive this method is not microscopy [80]. Additionally, interpreting the complex rami- practical for malaria diagnosis. fications of using multiple RDTs to diagnose P. knowlesi at the bedside is not practically feasible or economically viable. First Loop-mediated isothermal amplification Response® and ParaHIT® aRDTs were tested for their ability A PCR-based technology to harness specificity and sensitivity to identify P. knowlesi, P. falciparum and P. vivax infections [82]. but in a rapid and affordable format has been developed. Loop- However the sensitivity was poor for both aRDTs in identifying mediated isothermal amplification (LAMP) involves a complex P. knowlesi infections, particularly in patients with uncompli- mix of specifically engineered nucleic acid primers that combine cated disease [82]. Patients with P. knowlesi malaria are symp- specific binding to target DNA and self-binding to support loop tomatic at very low parasitaemia, therefore not only is priming in subsequent amplification rounds [88]. As a result specificity important but sensitivity is paramount [32,34]. Plas- large DNA structures and insoluble salts are formed in positive modium knowlesi replicates daily and can rapidly progress to life- reactions that allow for easy detection without the need for threatening hyperparasitaemia [8]. Any diagnostic test devel- specialized equipment [89]. oped for P. knowlesi must be specific and sensitive enough to A LAMP assay for species-specific rapid diagnosis of Plas- ensure early diagnosis, especially in the rural setting [34]. modium species has been developed [90]. LAMP assays are

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