TREPAR 1555 No.

of Pages 12

Review
The Treatment of Plasmodium
knowlesi Malaria
Bridget E. Barber,1,2 Matthew J. Grigg,1,2 Timothy William,2,3,4
4_TD$IF]
Tsin W. Yeo,1,2,5,6 and Nicholas M. Anstey[1,2,*
Plasmodium knowlesi occurs across Southeast Asia and is the most common
Trends
cause of malaria in Malaysia. High parasitaemias can develop rapidly, and the
Plasmodium knowlesi is the common-
risk of severe disease in adults is at least as high as in falciparum malaria. est cause of malaria in Malaysia and
Prompt initiation of effective treatment is therefore essential. Intravenous arte- has been reported throughout South-
east Asia. With risk of high parasitaemia
sunate is highly effective in severe knowlesi malaria and in those with moder- and severe disease, prompt initiation of
ately high parasitaemia but otherwise uncomplicated disease. Both chloroquine effective treatment is paramount.
and artemisinin-combination therapy (ACT) are highly effective for uncompli-
Risk of severe disease is strongly
cated knowlesi malaria, with faster parasite clearance times and lower anaemia associated with parasitemia: 11-fold
rates with ACT. Given the difficulties with [6_TD$IF]microscope diagnosis of P. knowlesi, a and 28-fold more likely with parasite-
mias >20 [3_TD$IF]000/mL and >100 [7_TD$IF]000/mL
unified treatment strategy of ACT for all Plasmodium species is recommended
respectively.
in coendemic regions.
Intravenous artesunate is highly
Emergence of P. knowlesi Malaria efficacious for severe knowlesi
malaria, and in those with parasitemia
The simian parasite P. knowlesi was first identified in 1932 [1]; however, it was not recognized as >20 [3_TD$IF]000 parasites/mL and otherwise
an important cause of zoonotic malaria until 2004, when Singh et al. reported a large focus of uncomplicated disease.
knowlesi malaria in Sarawak, Malaysia, in patients previously misdiagnosed with the microscopi-
In the only randomized clinical trial to
cally near-identical Plasmodium malariae [2]. Notifications of human knowlesi malaria in Malaysia
date, chloroquine and artesunate-
have increased substantially since this time, with P. knowlesi now the most common cause of mefloquine were both highly effective
malaria in this country [3,4] and threatening the otherwise successful progress towards malaria for uncomplicated knowlesi malaria.
elimination [5]. The simian reservoir and mosquito vectors for P. knowlesi are found in Southeast Parasite clearance times were faster
with artesunate-mefloquine.
Asia, with over 500 million people theoretically at risk of infection [6]. Human infections have been
reported in all countries in Southeast Asia except Timor Leste and Laos, and in travellers Microscopy misdiagnosis of P. know-
returning from this region [6]. P. knowlesi infection can rapidly progress to high parasitaemias, lesi is common; a unified strategy of
and in adults the risk of severe disease is at least as high as that of falciparum malaria [7]. Thus, artemisinin-combination therapy is
therefore recommended for all Plasmo-
prompt diagnosis and initiation of effective treatment is essential for preventing morbidity and
dium species in coendemic regions.
mortality.

In this review we discuss the treatment of uncomplicated and severe knowlesi malaria,
1
highlighting the importance of a unified treatment strategy of artemisinin-based treatment for Menzies School of Health Research
and Charles Darwin University, PO
all Plasmodium species in knowlesi-endemic areas.
Box 41096, Casuarina 0810, Northern
Territory, Australia
2
Diagnosis of P. knowlesi Malaria Infectious Diseases Society Sabah-
Menzies School of Health Research
While microscopy is sensitive for the diagnosis of P. knowlesi in clinical disease, the mature
Clinical Research Unit, Kota Kinabalu
trophozoites and schizonts of P. knowlesi resemble those of P. malariae, and the two species 88586, Sabah, Malaysia
3
cannot be reliably differentiated [8]. Laboratory reporting of P. knowlesi as the more benign Queen Elizabeth Hospital Clinical
Research Centre, Kota Kinabalu
P. malariae has been associated with failure to recognize severe malaria and consequent
88586, Sabah, Malaysia
delayed initiation of parenteral therapy, with fatal outcomes [9]. In areas of significant endemicity 4
Jesselton Medical Centre, Kota
for P. knowlesi, parasites with the microscopic appearance of P. malariae should be reported as Kinabalu 88300, Sabah, Malaysia
5
Lee Kong Chian School of Medicine,
P. knowlesi and treated as such. In Sabah, Malaysia, it is likely that the inclusion of P. knowlesi in
Nanyang Technological University,
laboratory microscopy reporting has contributed to increased recognition and more timely 639798 Singapore

Trends in Parasitology, Month Year, Vol. xx, No. yy http://dx.doi.org/10.1016/j.pt.2016.09.002 1

© 2016 Elsevier Ltd. All rights reserved.
 TREPAR 1555 No. of Pages 12

6
administration of intravenous artesunate for severe knowlesi malaria, and to the recent reduction Communicable Disease Centre,
Institute of Infectious Diseases and
in P. knowlesi case-fatality rates [3]. Epidemiology, Tan Tock Seng
Hospital, 308433 Singapore
By microscopy, P. knowlesi may also resemble P. falciparum due to similarity between the ring
forms of the two species [8], and misdiagnosis as Plasmodium vivax, and vice versa, is also
[5_TD$IF]*Correspondence:
common [10]. In Sabah, where ‘P. malariae/P. knowlesi’ accounted for 76% of all microscopy- nicholas.anstey@menzies.edu.au
based malaria notifications in 2014, PCR-confirmed P. knowlesi accounted for 21% and 38% of (N.M. Anstey).
blood films diagnosed as P. falciparum and P. vivax, respectively [11], highlighting the limitations
of microscopy for species differentiation in regions where P. falciparum, P. vivax, and P. knowlesi
all commonly occur.

Rapid diagnostic tests (RDTs) are insensitive for P. knowlesi malaria, particularly at low parasite
densities, and in general are unable to distinguish P. knowlesi from P. vivax [12,13]. Thus, while
microscopy remains the standard diagnostic method for clinical purposes, treatment decisions,
guidelines, and surveillance must take into account the potential inaccuracies of microscopy,
and molecular methods such as PCR are required to confirm the diagnosis of knowlesi malaria.
Confirmation of species by PCR will also allow administration of the antihypnozoite drug
primaquine to patients with P. vivax (or P. ovale) malaria, who may have been misdiagnosed
by microscopy as P. knowlesi [10].

In vitro Sensitivity of P. knowlesi to Antimalarials

In the only study to date evaluating ex vivo drug susceptibility of P. knowlesi, clinical isolates were
obtained from Sarawak, Malaysia. Growth inhibition by artemisinin, artemether, artesunate,
dihydroartemisinin, chloroquine, and mefloquine was assessed by using a modified schizont
maturation assay [14]. However, this study included only six clinical isolates, and the stage
specificity of the drugs tested, an important confounder of susceptibility of nonfalciparum
Plasmodium, was not assessed [15,16]. This may explain, in part, why the synchronised
laboratory P. knowlesi H strain appeared more sensitive to chloroquine (median 50% inhibitory
concentrations [IC50] 3.2 nM, range 2.2–4.7 nM) than the nonsynchronised clinical isolates
(median IC50 23 nM, range 11–38 nM), although with the latter IC50s still well below the
100 nM threshold used to define P. falciparum chloroquine resistance [17]. Both the clinical
isolates and the H strain of P. knowlesi were reported to be highly sensitive to artemisinin and its
derivatives [14], with IC50s <2.2 nM, lower than reported IC50s of artemisinins against
P. falciparum [18–20]. P. knowlesi isolates were reported to be less sensitive to mefloquine
than to chloroquine or artemisinins [14].

Drug Resistance Mutations

If, as is likely, P. knowlesi [9_TD$IF]malaria occurs primarily as a zoonosis [21,22], drug-selected mutations
would not be expected to occur. In the six Malaysian clinical isolates above, Fatih et al. analysed
the P. knowlesi orthologues of the P. falciparum chloroquine-resistance transporter (CRT) and
multidrug resistance protein 1 (MDR1) genes, associated with susceptibility of P. falciparum to
chloroquine and mefloquine, respectively, and did not detect mutations associated with drug
resistance [14]. In keeping with this, Tyagi et al. evaluated the CRT and dihydrofolate reductase
(DHFR) sequences of 53 P. knowlesi clinical isolates from the Andaman and Nicobar islands, and
found all to be wild type, with the latter suggesting lack of drug pressure from pyrimethamine
[23]. These findings have been extended by Grigg et al., who analysed the DHFR sequences of
over 400 P. knowlesi isolates from Sabah, Malaysia, and demonstrated multiple polymorphisms
but no evidence of drug selection [24]. Similarly, Assefa et al. analysed 48 P. knowlesi isolates
from Sarawak, Malaysia, and found no evidence of drug-selected mutations in the CRT, MDR1,
DHFR, dihydropteroate synthase (DHPS), and kelch K13 genes [25], with the latter two
associated with P. falciparum resistance to sulfadoxine and artemisinin derivatives, respectively.
Although Pinheiro et al. reported dimorphism and polymorphism among P. knowlesi isolates in

2 Trends in Parasitology, Month Year, Vol. xx, No. yy

 TREPAR 1555 No. of Pages 12

the multidrug-resistance-associated protein MRP1 and the multidrug-resistance protein MDR2

genes, both members of the ATP-binding cassette transporter family of genes associated with
mefloquine resistance in P. falciparum [26], the functional implications in P. knowlesi are not
clear.

Antimalarial Agents for the Treatment of Uncomplicated P. knowlesi Malaria

Chloroquine
Until recently, P. knowlesi was widely misdiagnosed as P. malariae due to microscopic near-
identity [2,8]. As such, P. knowlesi in Malaysia was almost uniformly treated with chloroquine,
which was until recently the recommended treatment of P. malariae in Malaysia. Initial studies
demonstrated chloroquine to be highly effective. In the first report of a large focus of human
infections with knowlesi malaria, Singh et al. reported 92 patients with knowlesi malaria treated
with chloroquine (25 mg/kg over 3 days) at a district hospital in Sarawak, with primaquine given
at 24 and 48 h [2]. Ten patients also received a single dose of sulfadoxine/pyrimethamine.
Severity status was not reported, although one patient had a parasite count >100 000 parasites/
mL. Median parasite clearance time was 2.4 days (range 1–5 days) and no deaths were reported.

The efficacy of chloroquine for the treatment of uncomplicated knowlesi malaria was further
demonstrated in a prospective observational study involving adults at the same district hospital
[27]. Chloroquine was given for 3 days with primaquine at 24 and 48 h. Patient characteristics
and parasite clearance kinetics in this (and other studies reported from knowlesi-endemic areas)
are shown in Table 1. Chloroquine plus primaquine was found to be highly efficacious. Mean time
to 50% (PCT50) and 90% (PCT90) reduction of parasitaemia at presentation was 3.1 h and
10.3 h, respectively [27]. All patients had cleared their parasites by day 3, and all were negative
by PCR on days 7, 14, 21, and 28. Similar efficacy of chloroquine was demonstrated by Grigg
et al. in a randomized clinical trial involving children and adults with uncomplicated malaria at
three district hospitals in Northeastern Sabah, Malaysia [28] (Table 1). Parasite clearance times
with chloroquine in this study were not confounded by concurrent primaquine treatment, and
were slower (PCT50 and PCT90 6.3 h and 14.8 h, respectively); nevertheless all patients had
cleared their parasites by day 3, and there were no treatment failures during the 42 days of follow
up. In both studies chloroquine appeared less effective against ring and early [10_TD$IF]trophozoite stages
compared to late trophozoites, with an increasing proportion of ring-stage parasites post-
treatment despite reduction in overall parasitaemia, as assessed by microscopy. The initial
parasite reduction ratio was lower with chloroquine than with artesunate-mefloquine, with
the difference reflecting the higher frequency of a transient increase in parasitaemia with
chloroquine [28]. The safety of chloroquine in patients with uncomplicated malaria and parasite
counts >20 000 has not been adequately established [7].

Artemisinin-Combination Treatment (ACT)

A variety of oral artemisinin-combination therapies have been successfully used for the treatment
of uncomplicated knowlesi malaria, including artesunate-mefloquine [28], artemether-lumefan-
trine [7,29], and dihydroartemisinin-piperaquine [30].

The best data exist for artesunate-mefloquine, which was compared to chloroquine in the only
randomized clinical trial in knowlesi malaria reported to date [28]. In this study, artesunate-
mefloquine was highly efficacious, with faster parasite clearance than with chloroquine (Table 1).
PCT50 and PCT90 for artesunate-mefloquine were 3.4 and 8.9 h respectively, with 84% of
patients aparasitemic by 24 h compared to 55% of patients treated with chloroquine (Table 1).
The difference in parasite clearance was particularly marked for ring-stage parasites (PCT50
8.6 h and 13.8 h for artesunate-mefloquine and chloroquine, respectively, P < 0.01; and PCT90
11.7 hours and 18.9 hours, respectively, P < 0.0001). As with chloroquine, there were no early
or late treatment failures. These results are consistent with an earlier nonrandomized study

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Table 1. Series of Antimalarial Chemotherapy Used in the Treatment of Plasmodium knowlesi Malaria in Adults and Children in Endemic Areas
Trends in Parasitology, Month Year, Vol. xx, No. yy

Study design Number Per cent Median age Median (range) PCT50 (h), PCT90 (h), median Parasite reduction % negative Time to parasite Died Refs
of patients male (range) parasitaemia median ratio at 24 h at 24 h clearance (<5/mL),
(parasites/mL) (%, 95%CI) median (range)

Uncomplicated malaria

Chloroquine and Prospective 33 58 46 3 724 3.1 10.3 99.4 (97.0–99.9) 33 35.8 0 [27]
primaquine observational (1845–7 480) (range 2.8–3.4) (range 9.4–11.4) (30.8–51.2) h

Chloroquine Randomised 125 75 32 (7–85) 1329 6.3 14.8 97.5 (96.6–98.4) 55 24 (6–60) h 0 [28]
controlled trial (33–35 873) (95%CI 5.1–7.5) (95%CI 13.4–16.1) (95%CI 45.2–64.4)

Chloroquine Retrospective 16 50 9 (4–14) 2240 NR NR NR 6a 2 days [64]

(200–14 400) (range 1–5)a

Artesunate/ Randomised 115 81 33 (3–82) 1457 3.4 8.9 99.8 (99.7–100) 84 18 (6–48) h 0 [28]
mefloquine controlled trial (36–35 008) (95%CI 2.9–4.0) (95%CI 8.2–9.6) (95%CI 76.4–90.5)

Artemether Prospective 27 70 31 (17–70) 4066 NR NR NR 33a 2 days 0 [7]b

-lumefantrine observational (232–60 840) (range 1–3)a

Artemether Retrospective 6 100 30 NR NR NR NR 66a 1 day 0 [29]b

-lumefantrine (range 0–3)a

Oral quinine Retrospective 11 83 45 NR NR NR NR 12a 2.5 days 0 [29]b

(range 1–3)a

Severe malaria

Intravenous Prospective 36 75 55 (20–74) 100 995 NR NR NR 33 a

NR 0 [7]b[1_TD$IF]
artesunate observational (32–584 015)

Intravenous Retrospective 6 63 56 NR NR NR NR 50% 2 days 1 (17%) [29]b

artesunate (range 1–3)a

Intravenous Prospective 10 30 64 (36–73) NR NR NR NR NR NR 2 (20%) [27]

quinine observational

Intravenous Retrospective 16 69 55 4+ NR NR NR 30% 4 days 5 (27%) [29]

quinine (range 2–7)a

a
Based on daily routine hospital microscopy.
b
Supplementary data.
NR, not recorded.
 TREPAR 1555 No. of Pages 12

demonstrating efficacy of artesunate-mefloquine for uncomplicated malaria at a tertiary referral

hospital in Sabah [7].

Mefloquine is recommended by the WHO only as an artemisinin partner drug, with use of
mefloquine monotherapy discouraged [31]. Successful treatment of uncomplicated knowlesi
malaria with mefloquine monotherapy has been reported [32,33]; however, treatment failures
have been reported in rhesus macaques [34,35], and in a single human case of knowlesi malaria
with high parasitaemia [36]. However, as with other agents [3,9,37], the treatment failure in this[1_TD$IF]
latter case may relate to the administration of oral therapy for[12_TD$IF] a high parasitaemia [13_TD$IF]infection, rather
than the recommended intravenous therapy (see below).

One case of mefloquine-related psychosis and suicidal ideation was reported in the clinical trial of
artesunate-mefloquine vs. chloroquine [28]. Mefloquine has been used extensively for the
treatment of uncomplicated falciparum and vivax malaria and its neuropsychiatric effects have
been well documented, with serious events occurring in 1 in 1217 Asian adults [38]. While
artesunate-mefloquine is approved by the Malaysian Ministry of Health as an alternative ACT for
uncomplicated knowlesi malaria in Sabah, ACTs with less risk of serious adverse events are
available.

Although no randomised controlled trials have been reported to date, artemether-lumefantrine is

listed by the Malaysian Ministry of Health as the preferred ACT for the treatment of uncompli-
cated knowlesi malaria and is widely used. In a prospective nonrandomised observational study
at a tertiary referral hospital in Sabah, 27 patients with uncomplicated knowlesi malaria were
safely and effectively treated with artemether-lumefantrine; while parasite clearance kinetics
were not fully reported, all patients had cleared their parasites by day 3 (Table 1) [7]. In a previous
retrospective study at the same hospital, patients with uncomplicated knowlesi malaria treated
with artemether-lumefantrine had faster parasite clearance times than those treated with
chloroquine or oral quinine [29].

Use of dihydroartemisinin-piperaquine has been reported in a single case involving a patient with
uncomplicated knowlesi malaria in Kalimantan, Indonesia [30]. Parasitaemia fell from 1.25% to
<0.01% within 24 h, and was undetectable by day 3.

Other Agents
Atovaquone-proguanil, although not widely available in knowlesi-endemic areas, has been used
for the treatment of uncomplicated knowlesi malaria in returned travellers (Table 2), with rapid
recovery reported in all cases [39–42].

Rationale for Unified Treatment Strategy of ACT for All Uncomplicated

Malaria
While chloroquine remains extremely efficacious for the treatment of uncomplicated knowlesi
malaria, artesunate-mefloquine is associated with faster fever-clearance time, faster parasite-
clearance time, reduced risk of post-treatment anaemia, and, in Malaysia, reduced bed-occu-
pancy due to the Ministry of Health policy of hospitalising patients until they have negative blood
films on two consecutive days [28]. For these reasons, malaria treatment guidelines in Malaysia
have recently been updated to recommend artemether-lumefantrine as first-line asexual-stage
treatment for uncomplicated malaria due to P. knowlesi and all other Plasmodium species.

The major rationale behind a unified ACT regimen for all species in coendemic regions is the
superior efficacy of ACT over chloroquine against multidrug-resistant P. falciparum and P. vivax
in this region [43]. Use of chloroquine for the treatment of microscopy-diagnosed knowlesi
malaria is associated with a risk of administering chloroquine to misdiagnosed P. falciparum or

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6

Table 2. Published Reports of the Treatment and Outcomes of Plasmodium knowlesi Malaria in Nonimmune Travellersa[2_TD$IF]

TREPAR 1555 No. of Pages 12

Trends in Parasitology, Month Year, Vol. xx, No. yy

Country of Region where infection Age/sex Parasitaemia Severity statusb Treatment Outcome/comments Refs
residence acquired

France Ko Payam, West 45 M 0.8% Uncomplicated Chloroquine Recovered [68]

Thailand

Sweden Bario Highlands, 35 M 0.1% Uncomplicated Mefloquine Recovered. Discharged afebrile after [32]
Sarawak, Malaysia 2 days

United States Palawan, Phillipines 50 F 2.9% Not stated Atovaquone-proguanil and Recovered [69]
primaquine

Singapore Perak, West Malaysia 11 M Not stated Uncomplicated Oral quinine, changed to Recovered. Discharged home after [70]
chloroquine 5 days

Australia Kalimantan, Indonesia 39 M 185 parasites/mL Uncomplicated Atovaquone-proguanil Recovered. Afebrile by 48 h [40]

New Zealand Bintulu, Sarawak, 40 M Not stated Uncomplicated Atovaquone-proguanil, changed Recovered [41]
Malaysia to artemether-lumefantrine

Finland West Malaysia 53 M <1% Uncomplicated Intravenous quinine, followed by Recovered. Treatment complicated [71]
oral quinine and doxycycline by hypoglycaemia, and an episode of
mild visual and hearing loss

Taiwan Palawan, Phillipines 60 M Not stated Uncomplicated Chloroquine Recovered [72]

c
Spain Southeast Asia (likely 39 M 250 parasites/mL Uncomplicated None Recovered [73]
West Malaysia)

Japan West Malaysia 35 M 10 120 parasites/mL Uncomplicated Mefloquine Recovered. PCT 40 h, FCT 28 h. [33]
Discharged on day 7; no relapses
during 5 months follow-up

Netherlands Kapit, Sarawak, 38 M 84 000 parasites/mL Severe (bilirubin 99 mmol/L with Chloroquine Recovered. PCT 40 h [46]
Malaysia parasite count >20 000/mL)

Netherlands Borneo, Malaysia 32 F 0.0005% Uncomplicated Atovaquone-proguanil Recovered. PCT 3 days [74]

Germany Ranong Province, 42 M 920 parasites/mL Uncomplicated Atovaquone-proguanil Recovered. Discharged on day 4 [42]
Thailand

Germany Ranong Province, 52 F 0.01% Uncomplicated Artemether-lumefantrine Recovered. Discharged on day 3 [75]
Thailand

Germany Myanmar or Thailand 73 M 3% Severe (ARDS, shock, acidosis, Intravenous quinine and oral PCT 48 h. Admission further [49]
renal failure, jaundice) doxycycline complicated by ventilator-associated
pneumonia. Required haemodialysis
for 5 weeks. Discharged after 5 weeks

Germany Phuket, Thailand 54 M 473 parasites/mL Uncomplicated Atovaquone-proguanil Recovered. Parasitaemia 11 [39]
parasites/mL at 48 h. Patent also
newly diagnosed with HIV

Germany Southern Thailand 55 F 0.2% Severe (acute kidney injury Intravenous artesunate then Recovered. PCT 2 days, creatinine [50]
developed following admission) artemether-lumefantrine normalised by day 4

a
Abbreviations: ARDS, acute respiratory distress syndrome; F, female; FCT, fever clearance time; M, male; PCT, parasite clearance time.
b
Based on clinical and laboratory data provided in the case reports.
c
Patient had taken prophylaxis with mefloquine and atovaquone-proguanil. Chloroquine given after PCR results revealed P. knowlesi, 2 months after discharge from hospital.
 TREPAR 1555 No. of Pages 12

P. vivax. Misdiagnosis of knowlesi malaria by microscopy is common, with P. knowlesi frequently

misdiagnosed as P. vivax or P. falciparum, and vice versa [10]. Chloroquine is ineffective for the
treatment of falciparum malaria throughout Southeast Asia, and chloroquine-resistant P. vivax is
also highly prevalent in this region, particularly in Indonesia and Malaysia [44,45]. In a recent
clinical trial involving 103 Malaysian adults and children with vivax malaria, the risk of recurrent
parasitaemia by day 28 was 61% in patients treated with chloroquine [44]. In contrast,
artesunate-mefloquine was highly effective, with rapid P. vivax clearance and no treatment
failures. This led to ACT being recommended over chloroquine as first-line treatment of
uncomplicated malaria from all Plasmodium species in Malaysia. A unified treatment strategy
of ACT for all uncomplicated malaria would be similarly advantageous in other regions coen-
demic for P. knowlesi, P. falciparum, and P. vivax, with the addition of primaquine radical cure for
P. vivax[14_TD$IF] (and P. ovale) malaria.

An additional advantage of a unified ACT treatment strategy is that it reduces the risk of
inadvertent chloroquine administration to patients with unrecognized severe knowlesi malaria.
Although there are isolated reports of successful use of chloroquine for the treatment of severe
knowlesi malaria [2,46], fatal outcomes have been reported with its use [9,37]. Although use of
oral ACT has been associated with a fatal outcome in a patient with a very high (>200 000/mL)
parasitaemia [3] and patients with severe malaria should receive intravenous artesunate (see
below), it is possible that the broader stage specificity and more rapid parasite clearance of oral
ACT may be associated with improved outcomes if used inadvertently in those with unrecog-
nised high parasitaemia.

Primaquine
P. knowlesi does not form hypnozoites and hence antirelapse therapy is not required. Sexual
stages are cleared rapidly with drugs used to treat asexual-stage infection, without use of
primaquine [28]. P. knowlesi gametocytes are cleared with nonprimaquine antimalarial agents.
The proportion of patients with gametocytemia, as evaluated by qPCR, is reduced from 85% to
6% and 4% 7 days after treatment with chloroquine and artesunate-mefloquine respectively,
with transmissibility of these residual gametocytes likely to be limited [28]. With P. knowlesi
existing primarily as a zoonosis without evidence for substantial human–human transmission
[21,22], administration of primaquine as a transmission-reducing agent in endemic regions is not
considered necessary, particularly in areas where testing for glucose-6-phosphate dehydroge-
nase (G6PD) deficiency is not available. Primaquine is indicated in cases where confirmatory
PCR identifies a true P. vivax infection misdiagnosed as P. knowlesi.

Treatment of Severe P. knowlesi Malaria

High parasitaemias can develop rapidly following P. knowlesi infection and lead to the com-
plications of severe malaria. Thus, prompt recognition of severe malaria and initiation of
appropriate treatment is essential for preventing morbidity and mortality. Severe knowlesi
malaria has been defined according to modified criteria for severe falciparum malaria
(Box 1), with a lower parasite count of 100 000 parasites/mL used as the cut-off to define
hyperparasitaemia [7,27]. In adults, the most common manifestations of severe knowlesi malaria
include acute kidney injury, hyperparasitaemia, jaundice, shock, and respiratory distress [6].
Metabolic acidosis occurs less commonly but has been reported in most fatal cases [3,9,37]. In
contrast to falciparum malaria, coma does not appear to be a feature of severe knowlesi malaria,
with no case of coma, with exclusion of other causes, yet reported in PCR-confirmed knowlesi
malaria. Also in contrast to falciparum malaria, severe disease from P. knowlesi has not been
reported in children, and no paediatric deaths have been reported [3]. Nearly all cases of severe
knowlesi malaria have been reported from Malaysia; however, severe knowlesi malaria has also
been reported in Thailand [47] and Brunei [48], and in travellers returning from endemic regions
[46,49,50] (Table 2).

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 TREPAR 1555 No. of Pages 12

Box 1. Indications for Parenteral Therapy with Artesunate in Plasmodium knowlesi Malaria
 Inability to tolerate oral intake
 Parasite count >20 000/mL and unable to assess other clinical and/or laboratory criteria for severe malaria
 Severe malaria (Table I)

Table I. Criteria for Severe Plasmodium knowlesi Malaria

Unrousable comaa Glasgow Coma Scale <11

Respiratory distress Oxygen saturation <92% with respiratory rate >30 breaths/min

Shock Systolic blood pressure <80 mmHg with cool peripheries or impaired capillary refill

Jaundice Bilirubin >50 mmol/L, with parasitaemia >20 000/mL and/or creatinine >132 mmol/L

Severe anaemia Haemoglobin <7.0 g/dL (adults)

Haemoglobin <5.0 g/dL (children)a

Significant abnormal bleeding –

Hypoglycaemia Blood glucose <2.2 mmol/L

Metabolic acidosis Bicarbonate <15 mmol/L or lactate >5 mmol/L

Acute kidney injury Creatinine >265 mmol/L

Hyperparasitaemia Parasite count >100 000/mL (or >2% or infected red blood cells)

a
Not reported to date in knowlesi malaria.

The WHO recommends treatment with intravenous artesunate for severe malaria of any species
[31], based on large randomized controlled trials in Southeast Asian adults and children [51] and
African children [52] demonstrating reduced mortality with intravenous artesunate compared to
intravenous quinine for the treatment of severe falciparum malaria. Intravenous artesunate has
also been shown to be highly effective for the treatment of severe knowlesi malaria. In a
prospective study at a tertiary referral hospital in Sabah, intravenous artesunate was adminis-
tered to 38 patients with severe knowlesi malaria, with zero mortality [7]. In an earlier retrospec-
tive study conducted at the same hospital, before and after the introduction of intravenous
artesunate in 2010 [29], 5 of 16 (31%) patients with severe knowlesi malaria treated with
intravenous quinine died, compared to 1 of 6 (17%) patients treated with intravenous artesunate.
In another prospective observational study in Sarawak, 2 of 10 (20%) patients with severe
knowlesi malaria and treated with intravenous quinine died [27]. This difference in case-fatality
rates is reflected in a recent state-wide study in Sabah, demonstrating that the fatality rate from
knowlesi malaria fell from 9.2/1000 case notifications in 2010 to 1.6/1000 case notifications in
2014 [3]. It is likely that this reduction in mortality is due at least in part to the increasing use of
intravenous artesunate for severe malaria.

While intravenous artesunate should clearly be administered to patients with clinical or laboratory
evidence of severe knowlesi malaria, and to those not tolerating oral medications (Box 1), the
optimal treatment for patients with moderately high parasite counts in the absence of other
evidence of severe disease is less certain. In a recent prospective study, 53% of patients with a
parasite count of >20 000 parasites/mL had severe disease, compared to only 9% of patients
with a parasite count of <20 000 [7]. The risk of severe knowlesi malaria increased 11-fold and
28-fold with parasitaemias >20 000 parasites/mL and >100 000 parasites/mL, respectively [7]. In
another study, there was a 64% chance of severe disease in patients with a parasite count above
35 000 parasites/mL [53]. In the randomized controlled study by Grigg et al., a small number of
patients with parasite counts of between 20 000/mL and 35 000 parasites/mL were safely treated
with both chloroquine and mefloquine-artesunate [28]. However, this small number of patients
did not allow confirmation of the safety of oral therapy at this level of parasitaemia. Thus, the
requirement for intravenous therapy for patients with parasite counts of 20 000–100 000/mL
in the absence of other clinical or laboratory evidence of severe disease remains uncertain. In

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 TREPAR 1555 No. of Pages 12

recognition of this uncertainty, the WHO recommends that patients with knowlesi malaria and
parasitaemia >20 000/mL receive intravenous artesunate if clinical or laboratory criteria for severe
disease cannot be rapidly or adequately assessed [54].

For patients fulfilling criteria for severe knowlesi malaria, intravenous artesunate should generally
be given for a minimum of 3 doses (2.4 mg/kg), 12 h apart [54]. This should be followed by a
complete course (3 days) of oral ACT such as artemether-lumefantrine once oral intake is
tolerated, according to local guidelines and availability. For patients commenced on intravenous
artesunate in the absence of criteria for severe malaria, oral ACT should be substituted as soon
as oral intake is tolerated.

In severe falciparum malaria intravenous artesunate has in some cases been associated with
post-treatment haemolysis, presenting as severe anaemia 7 to 14 days after treatment [55,56].
While early haemolysis with haemoglobinuria has been reported following artesunate use in
severe knowlesi malaria [57], post-artesunate delayed haemolysis has not yet been reported in
P. knowlesi infection. WHO recommends that all patients who receive intravenous artesunate be
monitored for delayed haemolytic anaemia [31].

Adjunctive and Supportive Therapy

There have been no clinical trials of adjunctive therapy in severe knowlesi malaria. However, [15_TD$IF]no
adjunctive therapies have proven effective in severe falciparum malaria, and none are recom-
mended for use in severe malaria from either species [54]. Severe knowlesi malaria is frequently
associated with multiorgan failure requiring intensive supportive management. Supportive
treatment for severe knowlesi malaria should be according to management guidelines for severe
falciparum malaria, including inotropic and ventilatory support [29], haemodialysis for acute
kidney injury [7,29], and blood transfusions as required [31,54].Thrombocytopenia is universal in
severe knowlesi malaria and significant abnormal bleeding, although uncommon, has been
reported [7,37,48,58,59]. Platelet counts recover rapidly after commencement of antimalarial
treatment [7]. While platelet transfusions have been given [16_TD$IF]for bleeding associated with severe
thrombocytopenia, the clinical benefits are uncertain. There have been no clinical trials to guide
intravenous fluid management in knowlesi malaria. In severe falciparum malaria liberal adminis-
tration of intravenous fluids has been shown to be deleterious [60,61] and conservative fluid
regimens have been shown to be safe [62]. Because the risk of acute respiratory distress
syndrome and noncardiogenic pulmonary oedema is at least as high in severe knowlesi malaria
as in severe falciparum malaria [29,54], careful use of conservative intravenous fluid regimens
would appear prudent [60].

There are no clinical trials to guide the use of empirical antibiotics in severe knowlesi malaria.
Concurrent bacteremia, predominantly Gram negative, occurs in up to 13% of adults with severe
falciparum malaria [62] and Enterobacter bacteremia has been reported in two series of severe
knowlesi malaria [7,29]. Empirical intravenous broad-spectrum antibiotics are commonly admin-
istered in severe knowlesi malaria with multiorgan failure until blood cultures are negative [7].

Treatment of P. knowlesi Malaria in Children

Although Plasmodium knowlesi is the most common cause of childhood malaria in Malaysia [3],
prospective studies reporting the clinical features of knowlesi malaria in children are yet to be
published. However, while mild to moderate anaemia and thrombocytopenia are common, the
multiorgan failure that characterises severe disease in adults does not appear to occur in
children, nor the severe anaemia commonly seen with falciparum and vivax malaria in this
age-group [63]. Indeed, there have been no cases of severe paediatric PCR-confirmed knowlesi
malaria reported to date [6,64]. The treatment recommendations for knowlesi malaria in children
are the same as for adults. In the randomized controlled trial by Grigg et al., 12 and 8 children

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 TREPAR 1555 No. of Pages 12

(defined as <12 years old) were treated with artesunate-mefloquine and chloroquine, respec- Outstanding Questions
tively [28]. Median (range) parasite counts in these children were 377 (36–9998) parasites/mL What is the threshold parasitaemia that
and 2024 (459–20 750) respectively, and all were aparasitaemic by 48 h [28]. The use of requires administration of intravenous
artesunate in patients with otherwise
chloroquine in an additional 16 children with uncomplicated knowlesi malaria was reported in uncomplicated knowlesi malaria?
a retrospective study; median parasite clearance time was 2 days, although a parasite
clearance time of 5 days was reported in an 8-year-old boy with an admission parasitaemia Should this threshold be lower in those
of 14 400 parasites/mL [64]. Successful use of oral and intravenous quinine for knowlesi malaria aged >50 years?
in children was also reported [64]. In the absence of reported severe knowlesi malaria in children,
What is the optimal artemisinin-combi-
the use of intravenous artesunate for this indication has not been described, but would be
nation therapy for uncomplicated
recommended based on its known efficacy in children with severe falciparum malaria [51,52], knowlesi malaria?
and in adults with severe knowlesi malaria [7].
When is it safe to administer antimalar-
Treatment of P. knowlesi Malaria in Pregnancy ial therapy for knowlesi malaria without
hospitalisation?
In contrast to P. falciparum and P. vivax, P. knowlesi infection during pregnancy appears to be
relatively rare, with only five cases reported to date [29,65]. However, despite its rarity, adverse
What are the indications for starting
maternal and infant outcomes have been reported, including severe maternal malaria, foetal loss and stopping intravenous fluids in
and low birth weight [29,65], and effective treatment is essential. Any woman with severe malaria knowlesi malaria, and are conservative
in pregnancy should be treated with immediate intravenous artesunate, regardless of species fluid regimens safe (as has been shown
in falciparum malaria)?
and including those in the first trimester [31]. Women with uncomplicated knowlesi malaria in the
second or third trimester of pregnancy may be treated with oral ACT, as per the guidelines for
What are the pathogenic mechanisms
treatment of uncomplicated falciparum malaria in pregnancy [31]. In the first trimester of in severe knowlesi malaria, and do
pregnancy uncomplicated knowlesi malaria may be treated with chloroquine. If there is any adjunctive therapies targeting these
doubt as to the microscopic diagnosis, alternative treatments include quinine and clindamycin, improve outcomes?

as per guidelines for falciparum and vivax malaria [31]. In falciparum malaria large studies have
found no increased risk of stillbirths, miscarriage, or major congenital malformations with
artemisinin regimens compared to nonartemisinin regimens [66,67]. Accordingly the WHO
Malaria Policy Advisory Committee has recommended that WHO update their 2015 Guidelines
to consider the timely inclusion of ACT as a first-line therapeutic option for falciparum malaria in
the first trimester of pregnancy, and artemether-lumefantrine may also therefore be an alternative
therapy for microscopy-diagnosed first trimester knowlesi malaria where there is doubt about
the diagnosis.

Concluding Remarks
P. knowlesi [9_TD$IF]malaria occurs primarily as a zoonosis[17_TD$IF], and [18_TD$IF]appears therefore free from drug-
resistance mutations developing as a result of human antimalarial use. Thus, while prompt
administration of intravenous artesunate is mandatory for severe disease, a range of antima-
larials is available for uncomplicated knowlesi malaria. Chloroquine remains highly effective for
uncomplicated disease. However, artemisinin-based treatments are associated with a superior
early therapeutic response and less anaemia. Moreover, treatment of uncomplicated knowlesi
malaria must take into account the possibility of misdiagnosed chloroquine-resistant P. falci-
parum or P. vivax, or unrecognized high parasitaemia, all of which would not be adequately
treated with chloroquine. Thus, ACT should be considered the treatment of choice for uncom-
plicated knowlesi malaria, and in areas coendemic for chloroquine-resistant P. vivax and
P. falciparum, there is a strong rationale for universal ACT for all species. The comparative
efficacy of the various ACTs has not been evaluated, and warrants prospective clinical efficacy
studies. In addition, given the strong correlation between parasite density and development of
complications in knowlesi malaria, further studies are required to determine the safety of oral
ACT in patients with moderately high parasitaemia (20 000–100 000/mL) but otherwise uncom-
plicated disease (see Outstanding Questions).

[19_TD$IF]Acknowledgments
National Health and Medical Research Council of Australia (Fellowships to BEB, TWY, NMA; Scholarship to MJG).

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 TREPAR 1555 No. of Pages 12
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