Review TRENDS in Parasitology Vol.20 No.
5 May 2004
Mixed-species malaria infections in
humans
Mayfong Mayxay1,2, Sasithon Pukrittayakamee3, Paul N Newton1,4
and Nicholas J White1,3,4
1
Wellcome Trust– Mahosot Hospital –Oxford Tropical Medicine Research Collaboration, Microbiology Laboratory,
Mahosot Hospital, Vientiane, Lao PDR
2
Faculty of Medical Sciences, National University of Laos, Vientiane, Lao PDR
3
Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok, 10400, Thailand
4
Centre for Clinical Vaccinology and Tropical Medicine, Churchill Hospital, Oxford, OX3 7LJ, UK
Mixed-species malaria infections are often not recog-
nized or underestimated. In Asia, surveys usually report
that < 2% of infections are mixed, whereas therapeutic
studies in vivax or falciparum malaria have demon-
strated a high prevalence (up to 30%) of infection with
the other malaria species during convalescence, sug-
gesting covert co-infection. In epidemiological studies,
a high prevalence of cryptic mixed-malaria species
infection has been detected by sensitive PCR tech-
niques. Concurrently infecting malaria species are
mutually suppressive with Plasmodium falciparum
tending to dominate Plasmodium vivax, but P. vivax
attenuating the severity of P. falciparum. There is evi-
dence for some cross-species immunity. These inter-
actions have important clinical and public health
implications.
As you read this article, ,6 – 10% of the world population
harbour malaria parasites in their bloodstream. Despite
over a century of attempts to control malaria, the disease
continues to cause high morbidity and mortality, and kills
between one and five people every minute. Several factors
contribute to this failure, including the spread of drug-
resistant Plasmodium falciparum parasites, anopheline
mosquito resistance to insecticides, socio-economic and
political factors, and the lack of access for the majority of
the world population to effective, inexpensive antimalarial
drugs. In addition, despite over a century of research,
much of the biology of malaria parasites and how they
interact with their human host and with each other
remains to be discovered.
Of the four species of malaria parasites that infect
humans (P. falciparum, Plasmodium vivax, Plasmodium
malariae and Plasmodium ovale), P. falciparum and
P. vivax are the most common. Plasmodium falciparum
causes the most severe disease and almost all fatalities,
whereas P. vivax, although usually considered to be
benign, causes repeated debilitating relapses, rare life-
threatening complications [1], and low-birth-weight
(LBW) infants of infected mothers [2]. LBW also increases
the risk of death in the newborn.
Corresponding author: Nicholas J White (nickw@tropmedres.ac).
www.sciencedirect.com 1471-4922/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2004.03.006
Mixed infections
Even in areas of low transmission, a high proportion of
malaria infections is with more than one genotype from a
particular species. Because several malaria parasite
species of humans are usually present in a particular
area, infections with more than one species of Plasmodium
at the same time are also expected. Indeed, these were
described soon after the identification of malaria parasites
of humans and their speciation, but there has been only
sporadic interest in this aspect of malariology until
recently. Infections with multiple pathogens, transmitted
by the same vector or route, have been described in other
infections. For example, mixed infections with Lyme
disease, human granulocytic ehrlichiosis and babesiosis,
transmitted by Ixodes ticks occur together in northeastern
USA [3], and positive associations between geohelminths
are common globally [4].
In clinical practice, mixed malaria infections are often
not recognized. Mixed infections diagnosed by microscopy
in patients admitted to hospital and in epidemiological
surveys are a small proportion (, 2%) of the total
prevalence [5,6]. This represents both observer error,
difficulty in distinguishing the young ring-form parasites
of the four malaria species of humans, the undetectable
presence of P. vivax and P. ovale liver hypnozoites, and that
many infections are at densities below the threshold of
detection by microscopy (, 1 £ 108 parasites in an adult).
However, longitudinal studies of patients who have not
been re-exposed to malaria after the initial malaria
attack have demonstrated that mixed infections are
remarkably common, with the delayed appearance of
one cryptic species, following treatment for the species
which was patent at presentation [5,7]. For example, in
Thailand, over one-third of patients presenting with acute
P. falciparum malaria harbour cryptic P. vivax simul-
taneously [5]. Conversely, , 8% of patients presenting with
P. vivax malaria harbour cryptic P. falciparum simul-
taneously [8]. If antimalarial treatment does not eradicate
the cryptic species, it often becomes patent during
convalescence.
In support of these observations, recent work using
sensitive PCR detection of parasite DNA has demon-
strated a much higher prevalence of mixed infections than
234 Review TRENDS in Parasitology
appreciated from microscopy [9,10]. Failure to detect
mixed infections could result in inadequate or incorrect
treatment. A missed diagnosis of P. falciparum within a
mixed infection could potentially result in severe disease.
Plasmodium species interactions
The classical experiments in humans conducted before the
Second World War [11 –13] showed reciprocal numerical
dominance of one species over the other after simultaneous
inoculation of P. falciparum with P. vivax, and P. vivax with
P. malariae. These experiments suggested that different
malaria parasites mutually interact or antagonize each
other in the infected host. Jeffery [14] found no cross-
protection between P. falciparum and P. vivax, but found
evidence that P. ovale ameliorated subsequent P. falci-
parum infections.
That malaria species interact has been supported by
subsequent field observations. First, the prevalence of
mixed infections in cross-sectional malaria surveys is less
than would be expected from the prevalence of single-
species infections [15]. Second, there is reciprocal season-
ality in the prevalence of different species [16]. For
example, in Vanuatu, P. vivax and P. falciparum demon-
strate a striking reciprocal seasonality, not attributable to
mosquito vector behaviour or abundance, or to drug use –
P. falciparum infection appears to suppress P. vivax
parasitaemia [17]. In recent studies from Papua New
Guinea, it was shown that over a period of 61 days, 82% of
semi-immune children harboured more than one species of
Plasmodium and that peaks of parasitaemia followed a
sequential pattern without concurrent high parasitaemias
of more than one species. The data were consistent with
inter-species interactions that were density-dependent
resulting in a relatively stable total Plasmodium parasit-
aemia [18]. Third, longitudinal studies and PCR studies
have demonstrated that cryptic mixed-infections are com-
mon [5,6,8,9]. Fourth, there is evidence from Sri Lanka that
the severity of malaria symptoms is reduced in individuals
with limited pre-exposure to different species [19].
Although malaria parasites appear to engage in mutual
suppression, which species suppresses which remains
controversial. Many studies suggested that P. falciparum
suppresses P. vivax, although the reverse might also occur
[8]. In a recent analysis of 73 datasets of infections with
multiple Plasmodium spp., little consistency was found; of
22 significant associations between P. falciparum and
P. malariae, 14 were positive, and of 26 associations
between P. falciparum and P. vivax, 17 were negative.
Associations between malaria species tended to be positive
in Africa and negative in Asia [4]. Which species
suppressed which probably depends on factors such as
the relative rates of replication, the timing of inoculation,
competition for nutrients and red blood cells, and through
the host immune response, either through early activation
of non-specific host defence mechanisms or via hetero-
logous immunity [20]. Whereas P. vivax infects only young
red blood cells up to 14 days after leaving the marrow
(Simpson et al. 1999 as cited in Ref. [1]), P. falciparum,
although preferring younger red blood cells, can also infect
older red blood cells [1]. Hence, dyserythropoiesis caused
by P. falciparum infection reduces the availability of red
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Vol.20 No.5 May 2004
blood cells for P. vivax invasion. Activation of non-specific
host-defence mechanisms (e.g. augmentation of splenic
clearance by the inflammatory response, fever) could limit
multiplication of the accompanying parasite – particu-
larly at the stage of exponential growth. Cross-species
immunity has been demonstrated experimentally in
simian and murine malaria species. In human malaria,
there are suggestions from clinical studies (Maitland et al.,
1996 as cited in Ref. [17]) [19] and there is in vitro evidence
for short-lived heterologous immunity to the toxin from
vivax and falciparum malaria parasites. Maitland et al.
[17] suggest that the cross-species immunity is likely to be
antitoxic, rather than antiparasitic in character. Inhi-
bition of rosetting by immune sera shows some cross-
species activity. Heterologous immunity between pairs
of parasite species might not be symmetrical with, for
example, P. ovale being less susceptible to anti-P. falci-
parum immunity than vice versa.
Geographical distribution of mixed infection
The characteristics and distribution of malaria species,
anopheline mosquitoes, the individual host and their
interaction determine the spectrum of mixed infections.
The global distribution of malaria species is uneven:
P. falciparum is predominant in Africa, Papua New
Guinea and Haiti, whereas P. vivax is more common in
Central and parts of South America, North Africa, the
Middle East and the Indian sub-continent. The prevalence
of both species is approximately equal in other parts of
South America, East Asia and Oceania. Plasmodium vivax
is rare in sub-Saharan Africa, whereas P. ovale is rare
outside West Africa. Plasmodium malariae is found in
most areas, but is relatively uncommon outside Africa. In
Thailand, as in many countries where both P. falciparum
and P. vivax are found in approximately equal proportions
(Luxemburger et al., 1996 as cited in Ref. [21]), when the
incidence of one species rises, the reported incidence of the
other falls; whether this represents a true reciprocal
relationship or is an artifact of reporting is not clear.
Anopheline mosquitoes
At least seven species of Anopheles have been shown to
carry more than one species of human Plasmodium in the
field [6]. For example, mixed P. falciparum and P. vivax
were simultaneously found in Anopheles dirus [22],
P. falciparum, P. vivax and P. malariae in Anopheles
maculatus [23], and all four malaria species of humans can
be carried by Anopheles gambiae [24].
Because female anopheline mosquitoes sample human
blood repeatedly during their lives, different Plasmodium
spp. and different genotypes within species could accumu-
late. A higher prevalence of mixed-species infections would
be expected in older than in younger mosquitoes [6].
However, in areas of low transmission, such repeated
sampling will be unusual and presumably most mixed
infections in mosquitoes are due to simultaneous infection
by gametocytes of multiple species at one feed. In contrast
to the prevalence studies in humans, the frequency of
mixed species in mosquitoes occur at frequencies greater
than or equal to that predicted from the frequency of
single-species infections [6]. This could result from the
 Review TRENDS in Parasitology Vol.20 No.5 May 2004 235

under-recognition of mixed infections and that trans-
mission can occur at gametocytaemias slightly lower than
the level of microscopic detection. Comparison of the
prevalence of mixed-species infections in mosquitoes and
humans suggests little relationship [6], but the analysis is
bedeviled by different timings and locations of sampling of
mosquitoes and humans, and more sensitive assays for
Plasmodium in mosquitoes (enzyme immunoassays) than
in humans (slide microscopy). There is also evidence that
the anopheline mosquitoes co-infected with microfilaria
and Plasmodium retard the development of each other and
increase mosquito mortality [25].
Host factors
Intrinsic host factors might also determine the prevalence
of mixed infections. For example, people in west Africa
whose red blood cells lack the Duffy antigen will not
acquire mixed infections with P. vivax because Duffy-
negative red blood cells are resistant to infection with
P. vivax. It remains unclear whether thalassaemias and
human leukocyte antigen (HLA) polymorphisms, associ-
ated with altered risk for falciparum malaria, also
influence risk for acquiring other Plasmodium spp. [1].
Travelling to different malaria endemic areas could
contribute to mixed infections because a person could
acquire different Plasmodium spp. from different areas.
Frequency of mixed infections
The apparent frequency of mixed infections is dependent
on the methods used for parasite detection. Conventional
examination by microscopy at the time of the initial
diagnosis of malaria has a relatively low sensitivity for
multiple infections for at least four reasons: (i) it is difficult
to distinguish young ring-forms of the four Plasmodium
spp. by microscopy. (ii) The species might be present at
a low parasite density, beneath that of detection by
microscopy (i.e. , , 50 parasites ml21 blood). (iii) A micro-
scopist, on finding one species, might not search the slide
for a rare second species. (iv) The blood might be clear
of parasites, but hypnozoite stages persist in the liver
(P. vivax and P. ovale). Although experience of the
microscopist is important in identifying mixed infections,
even expert microscopists can misdiagnose low-density
mixed infections as single infections [8]. Therefore, the
overall proportion of mixed infections detected microscopi-
cally is underestimated. Mixed infections can be detected
by more sensitive PCR detection of species-specific DNA at
one time point, or through longitudinal follow up, outside
of a malaria-endemic area, of patients treated for an
apparent single-species infection.
PCR detection
In Thailand and in Laos, mixed infections detected on
admission and in epidemiological surveys using micro-
scopy have been reported as , 2% and ,1%, respectively
[26 –28]. This increases to , 55 – 65% when more sensitive
detection methods, such as PCR, are used (Table 1). As
expected, double-species mixed infections are more com-
mon than triple- and quadruple-species mixed infections.
Double-species infections can commonly be detected both
by microscopy and by PCR methods, whereas most triple-
and quadruple-species infections are usually identified by
PCR and are very infrequent (,5%) [29 – 32] (May et al.,
1999 as cited in Ref. [30]).
Longitudinal studies
Studies with long-term follow up of patients who presented
with malaria with one parasite species upon the admission
blood film examination have demonstrated a high preva-
lence of patent infection with another species during
convalescence (Tables 2 and 3). This method will also
underestimate the true prevalence of mixed infections
because it relies on treatment failure or, in the case of
P. vivax or P. ovale, relapse because only some infections
with these parasites will be resistant to the antimalarial
treatment or will relapse. The majority of reports are from
Bangkok where patients were followed up in hospitals
outside the area of malaria transmission for $28 days,
excluding re-infection as a confounding factor [8]. In
Bangkok, ,30% of patients treated for falciparum malaria
develop subsequent patent P. vivax parasitaemia without
re-exposure [5]. This indicates that the patients harboured

Table 1. Mixed infections not detected by microscopy of admission blood slide, but detected by PCR techniquesa
Mixed infections tested Mixed infections tested Cryptic species Countries Refs
positive by microscopy (%) positive by PCR (%)
0/23 (0) 4/23 (17) Pv Australia [51]
1/137 (0.5) 9/173 (5) Pv Thailand –b
0/189 (0) 52/189 (27.5) Pm Guinea Bisseau [52]
1/196 (0.5) 25/196 (13) Pv; Pm Thailand –c
0/48 (0) 6/48 (12.5) Pf; Pv Thailand [53]
18/475 (4) 356/548 (65) Pf; Pv; Pm; Po Thailand [9]
0/100 (0) 17/100 (17) Pf; Pv Venezuela –b
11/159 (7) 44/159 (28) Pf; Pm; Po Equatorial Guinea [54]
2/192 (1) 9/192 (5) Pv; Pm; Po Spain –d
80/1470 (0.005) 113/173 (65) Pf; Pv; Pm; Po Papua New Guinea [55]
2/58 (3.4) 27/117 (23.1) Pf; Pv; Pm; Po Laos [28]
7/300 (2.3) 26/151 (17) Pf; Pv; Pm; Po Thailand [56]
0/160 (0) 21/160 (13) Pf Thailand [34]
a
At the presentation of the patients, mixed-species malaria infection is usually underestimated when detected microscopically, but this is more common when detected by the
more sensitive technique – PCR. The overall median (range) ratio of PCR to microscopy diagnosed mixed infection in this review was 6% (0– 25%). Please note that the results
from different papers might not be directly comparable. Abbreviations: Pf, Plasmodium falciparum; Pm, Plasmodium malariae; Po, Plasmodium ovale; Pv, Plasmodium vivax.
b
Data obtained from Postigo et al. (1998) and Brown et al. (1992) as cited in Ref. [40].
c
Data obtained from Snounou et al. (1993) as cited in Ref. [39].
d
Data obtained from Rubio et al. (1999) as cited in Ref. [54].

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236 Review TRENDS in Parasitology Vol.20 No.5 May 2004

both P. falciparum and P. vivax parasites at presentation,
but that the recurrence of P. vivax was a relapse from
persistent liver stages because blood-stage infection
should have responded to the acute treatment. However,
as only some 60% of infections relapse in Thailand, this
suggests a staggering 30 out of 60 (50%) of infections are
with the two malaria species. The timing of the appearance
of asexual P. vivax in the peripheral blood following
treatment for P. falciparum depends on the antimalarial
drugs used. A shorter interval (less than four weeks) was
observed in the patients treated with short half-life
antimalarial drugs, such as the artemisinin compounds
or quinine, and a longer interval (four to six weeks) in
patients treated with long half-life drugs, such as
mefloquine or chloroquine (CQ) [1]. The more rapid
appearance of P. vivax in patients treated with short
half-life drugs corresponds to the interval period of P. vivax
relapse in Southeast Asia [7,33] which is about three weeks
without hypnozoitocidal treatment. This suggests that
P. vivax appearance following treatment for P. falciparum
is due to relapse. The retarded appearance of P. vivax after
the clearance of P. falciparum in patients treated with long
half-life drugs suggests that the antimalarial drug
concentrations suppress asexual P. vivax relapse para-
sitaemia until they can become patent or cure the first, but
not the second, relapse [7,33]. Approximately 10% of
patients treated for P. vivax and followed up without
potential re-exposure to malaria had subsequent appear-
ance of P. falciparum. This figure has been confirmed by
PCR detection studies, which demonstrated ,10 – 13%
cryptic P. falciparum in blood samples from patients
diagnosed as having vivax malaria by microscopy [7,34].
The mean onset of P. falciparum appearance in most cases
is less than two weeks. Unlike P. vivax, P. falciparum

Table 2. Mixed infections with Plasmodium vivax detected following treatment for Plasmodium falciparum malariaa
Antimalarial treatment Mixed infections tested Onset of P. vivax appearance Refs
positive (%) after antimalarial treatment
(range) [SD]
Artemisinin derivatives alone or in combination
ATS 13/82 (16) 20 (15– 24) –d
ATS 23/80 (29) (12 –34) [57]
ATS 11/25 (44) (12 –28) [58]
ATS þ DOX 16/55 (29) (18 –29) –d
ATS þ MFQ 57/308 (18.5) Within 63 days of follow up – b,e
ATM 2/38 (5) 11 and 21 [61]; – c
ATM 9/46 (20) (14 –23) [63]
ATM 1/5 (20) 22 [59]
ATM 38/87 (44) (13 –32) –d
ATM þ LMF 54/359 (15) 32 (23– 43) [60]; – b
ATM þ LMF 83/309 (26.5) Within 63 days of follow up – b,e
ATS or ATS þ azithromycin or LMF 8/80 (10) (7 –23) [8]
ATM þ MFQ 0/44 No P. vivax appearance –d
ATM, ATS or QN 16/382 (4) ? [62]
MFQ or halofantrine, and MFQ combinations
MFQ 1/46 (2) 52 [63]
MFQ 46/145 (32) 47 (30– 65) [5]
MFQ 3/40 (7.5) 38 [5] or (33 –42) [64]
MFQ 43/118 (36) (29 –63) –f
MFQ 1/65 (1.5) 28 [65]
MFQ 12/82 (15) 36 [9] or (23 –45) [66]
MFQ 43/118 (36) (29 –63) –f
MFQ þ DOX 1/54 (2) 21 –d
Halofantrine 7/62 (11) (14 –28) [65]
QN and QN combinations
QN 12/55 (23) 21.6 [3] –g
QN 1/23 (4) ? [51]c
QN þ CQ 3/25 (11) (29 –51) [67]
QN þ CQ 3/35 (12) (51 –59) [67]
QN þ clindamycin 12/60 (20) 25.1 (1.4) –g
QN þ Fansidar 1/23 (4) 60 [51]; – c
QN þ TET 8/25 (33) 29 [9.7] or (21 –50) [67]
QN þ TET 13/46 (28) 23 (18– 47) –d
QN þ TET 9/48 (19) 24.8 [3.2] –g
QN þ TET 8/25 (33) (21 –50) [67]
QN þ TET 13/46 (28) 23 (18– 47) –d
QN þ TET 17/78 (18) 28 (15– 21) –d
a
The data extracted from the papers gave the average days in different ways, so these are shown as median (range) or mean [SD]. Abbreviations: ?, data not available; ATM,
artemether; ATS, artesunate; CQ, chloroquine; DOX, doxycycline; LMF, lumefantrine; MFQ, mefloquine; QN, quinine; SD, standard deviation; TET, tetracycline.
b
Studies conducted in endemic areas.
c
Studies conducted outside Thailand.
d
Data obtained from Meek et al. (1986) and Looareesuwan et al. (1997) as cited in Ref. [40].
e
Data obtained from van Vugt et al. (1998) as cited in Ref. [60].
f
Data obtained from Harinasuta et al. (1983) as cited in Ref. [64].
g
Data obtained from Pukrittayakamee et al. (2000) as cited in Ref. [7].

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 Review TRENDS in Parasitology Vol.20 No.5 May 2004 237

Table 3. Mixed infections with Plasmodium falciparum detected following treatment of Plasmodium vivax malariaa
Antimalarial treatment No. of mixed infections tested positive (%) Onset of P. falciparum appearance after antimalarial Refs
treatment (days) [SD]
CQ 6
Halofantrine 2
PQ 3
Overall 11/166 (7.0) 13.1 [5.7] or (5 – 21) –b
CQ 104/992 (10.5) 12.6 [6.8] or (1 – 28) [46]
ATS þ PQ 1 23
PQ 2 10, 25
SP 2 9, 12
SP þ PQ 1 7
Overall 6/66 (9.0) 14 [7.0] [68]
CQ 6/324 (2.0) Within 28 days [21]; – c
CQ þ PQ 10/258 (4.0) Within 63 days
CQ 2
PQ 3
Overall 5/60 (8.0) (11– 26) –b
ATM 2/40 (5) 11 [61]; – d
AMQ ? 14 [69]; – c,d
CQ 4 (2 –21)
CQ þ PQ 4 (17– 21)
PQ 7 (2 –9)
Azithromycin 6 (8 –19)
Halofantrine 1 13
SP 1 7
Overall 23/234 (10) 10.4 [6.1] or (2 – 21) [8]
CQ 104/994 (10.5) 12.6 [6.8] or (1 – 28) [70]
a
The data extracted from the papers gave the average days in different ways, so these are shown as median (range) or mean [SD]. Abbreviations: AMQ, amodiaquine; ATM,
artemether; ATS, artesunate; CQ, chloroquine; PQ, primaquine; SD, standard deviation; SP, sulfadoxine– pyrimethamine; ?, data not available.
b
Data obtained from Pukrittayakamee et al. 2000 as cited in Ref. [7].
c
Studies conducted in endemic areas.
d
Studies conducted outside Thailand.

has no hypnozoite stage. Therefore, the appearance of
P. falciparum after P. vivax treatment derives from
circulating erythrocytic asexual stages not killed by the
drugs used to treat P. vivax (usually CQ, which is not
effective against P. falciparum in Thailand and in most of
the rest of the world). A study in Thailand [8] also
suggested that the appearance of P. falciparum following
clearance of P. vivax might be due to difficulties in
differentiating the ring forms of P. falciparum and
P. vivax on the admission slide. An alternative explanation
is that the intra-hepatic pre-erythrocytic stage of
P. falciparum might be prolonged in a mixed infection.
Benefit of mixed infections to the host
The implications of mixed infections to the host remain
controversial. Some workers have suggested that mixed
infections are beneficial, whereas others have suggested that
they are detrimental to the host. In the 1930 s, clinical
studies of human malariotherapy for neurosyphilis demon-
strated that P. falciparum suppressed P. vivax parasitaemia
when both species were inoculated simultaneously [11,13].
Naturally acquired mixed infections with other less severe
malaria species appear to attenuate the severity of
P. falciparum infection [6,17,21,35] (Luxemburger et al.,
1996 as cited in Ref. [21]). For instance, in western Thailand,
patients in whom only P. falciparum was diagnosed were
more likely to develop severe disease (5.7%) than those
presenting with P. falciparum and P. vivax mixed infections
(1.6%) (Luxemburger et al., 1997 as cited in Ref. [21]).
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Patients co-infected with P. vivax were 1.8 (95% CI; 1.2–2.6)
times less likely to become anaemic than those with
P. falciparum only. Gametocyte carriage was also reduced
when compared with single infections with P. falciparum
(Price et al., 1999 as cited in Ref. [36]). On the Thai–Burma
border, pregnant women whose first attack of malaria during
pregnancy was caused by P. vivax had a significantly lower
risk of developing P. falciparum later in the pregnancy [2].
This evidence raises the possibility that if vivax, but not
falciparum, malaria is controlled, falciparum malaria
could become a more serious public health problem. This
is of relevance to empirical treatment. Most malaria is
treated on the basis of symptoms, without parasitological
confirmation. If CQ is used for the treatment of vivax
malaria in areas of high-level resistance to P. falciparum,
the reduction in prevalence of vivax infection might
therefore prevent P. vivax attenuating P. falciparum.
Thus, the severity of disease could be increased. At higher
levels of transmission, mixed infections with other species
might also protect against clinical disease resulting from
P. falciparum. African children with mixed infections
comprising P. falciparum and P. malariae and/or P. ovale
had fewer or no symptoms compared to those with single-
species infections in admission blood samples [29]. This is
consistent with the negative association between mixed
infections (P. falciparum and P. malariae) and fever [37].
Plasmodium vivax has a much lower pyrogenic density
than P. falciparum, but how pyrogenic density is altered in
mixed infections is not known [1].
238 Review TRENDS in Parasitology
Mixed infections with P. vivax are associated with lower
P. falciparum parasitemia compared with P. falciparum
single infections [29]. Co-infection with P. vivax is also
associated with significantly fewer P. falciparum geno-
types per infection [38]. In therapeutic trials, patients
with mixed P. vivax and P. falciparum infections had a
lower risk of treatment failure compared with those with
P. falciparum single infections (Price et al., 1997, as cited
in Ref. [36]). Children with mixed infections of P. falciparum
and P. malariae and/or P. ovale had higher antibody titres
compared with single infections [29], although the
significance of this observation is uncertain. Mathematical
modeling suggests that P. malariae can reduce the peak
parasitaemia of subsequent P. falciparum infection by 50%
and that P. vivax can maintain stable P. falciparum
parasitaemia during acute infections [39,40].
Interactions between Plasmodium spp. might also affect
the prevalence and infectivity of gametocytes. If parasite
production is diverted to non-pathogenic sexual stages, this
could be of benefit to the patient, but if this increased malaria
transmission, mixed-species infections might have an impor-
tant negative public health impact. McKenzie et al. [41]
analyzed the neurosyphilis malariotherapy data and found
that earlier and increased P. falciparum gametocyte pro-
duction was associated with prior or concurrent P. malariae
infection when compared with P. falciparum single infec-
tions. By contrast, in Papua New Guinea, the presence of
P. falciparum gametocytes in the peripheral blood was
associated with reduced infectivity of concurrently present
P. vivax gametocytes [42]. In western Thailand, the risk of
gametocyte carriage of P. falciparum was reduced 3.5-fold
when the infection was mixed with P. vivax (Price et al.,
1999 as cited in Ref. [36]). Hence, measures that pre-
ferentially target vivax malaria, such as use of CQ in areas
with CQ-resistant P. falciparum, could increase the trans-
mission of falciparum malaria. However, there is also evidence
that the presence of P. falciparum gametocytes reduces the
infectivity of P. vivax gametocytes in mixed infections [42].
Cross-species non-specific immune responses have
been suggested as an explanation for the attenuation of
P. falciparum disease severity by vivax co-infections.
Maitland et al. [17] proposed the existence of an antitoxic
cross-immunity between P. falciparum and P. vivax which
controls, rather than eliminates, the infection. They
suggested that infection with P. vivax might result in the
development of a cross-species immunity that ameliorates
the clinical course of subsequent P. falciparum infection.
Downregulation of cytokine cascades might also be import-
ant [37]. The recent observation that febrile temperatures
are associated with increased P. falciparum cytoadherence
in vitro [43] suggests the possibility that the lower body
temperatures found in patients with mixed infections
including P. falciparum might be associated with less
cytoadherence and hence the observed milder disease.
Disadvantages of mixed infections to the host
A minority of clinical studies suggests that mixed
infections with Plasmodium spp. could be associated
with more severe disease. In India, Gopinathan and
Subramanian [44,45] found that mixed infections with
P. falciparum and P. vivax were associated with cerebral
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Vol.20 No.5 May 2004
malaria. This has not been observed in large prospective
series in Thailand or Vietnam. Double infections
(P. falciparum and P. vivax) or triple infections
(P. falciparum, P. ovale and P. malariae) have been
associated with more severe anaemia compared with that
in a single infection [8,30,46]. There is also conflicting
evidence as to whether spleen size is increased or reduced in
patients with mixed infections compared with those with
single-species infections [15,47]. The main danger of missed
mixed infections is that the incorrect treatment could be
given. Missed falciparum malaria results in the risk of
developing subsequently severe, possibly fatal, disease.
Missed vivax and ovale infections could result in repeated
debilitating infections and their associated economic and
social consequences, and missed vivax malaria during
pregnancy could result in the birth of a baby with LBW.
An additional practical concern is that inapparent mixed
infection might confound comparisons between comparative
clinical trials of antimalarial drugs and in vivo investi-
gations of the pathophysiology of malaria with consequent
adverse influences on public health policy.
Diagnosis
The diagnosis of mixed malaria infections is difficult in
clinical practice. Recurrent fever following clearance of a
malaria species should not be assumed to represent a
treatment failure; subsequent appearance of another
species from cryptic mixed infections should be considered
and the blood film examined carefully to determine the
species of the parasite. Speciation can be difficult if the
circulating stages of both species are immature and if
there is doubt, P. falciparum parasites should be diag-
nosed. The P. falciparum histidine-rich protein-2 (HRP2)
dipstick test has been shown to be useful in identifying co-
infection with P. falciparum, in patients presenting with
apparently single-species vivax malaria: the sensitivity was
75% and specificity 89% [8]. This indicates that the sub-
patent P. falciparum parasite density was close to that of
detection by microscopy. Rapid dipstick tests based on the
detection of parasite lactate dehydrogenase enzyme can
detect both P. falciparum and non-P. falciparum parasites
(i.e. P. vivax, P. ovale and P. malariae) on the same strip [1].
Unfortunately, this test cannot distinguish between
P. falciparum infection only, and mixed infections with
P. falciparum and P. vivax. PCR techniques, although useful as
a research tool, are too expensive and cumbersome for routine
diagnostic use in the tropics, and are prone to contamination.
Treatment
There have been no specific recommendations for the
treatment of mixed infections and we have found only one
clinical study specifically addressing this issue [48]. The
immediate drug treatment for infections with P. falciparum
mixed with another malaria species should be the rec-
ommended treatment for falciparum malaria because
P. falciparum infection is potentially fatal. The optimum
therapeutic regimen will differ, based on the local sensitivity
pattern of P. falciparum [1]. In general, P. vivax is equally or
more susceptible to antimalarial drugs compared with that
by P. falciparum, with two potential exceptions. In a small
cohort study conducted in an area of the Amazon without
 Review TRENDS in Parasitology
malaria transmission, five days of clindamycin cured
patients with a single infection with P. falciparum, but
patients with P. falciparum plus P. vivax infections had
longer parasite clearance times and all had P. vivax
recrudescence or, less likely, hypnozoite relapse three to
four weeks after chemotherapy [48]. Sulfadoxine– pyri-
methamine (SP) should not be used alone in the treatment
of mixed infections including P. vivax because vivax
parasites rapidly develop resistance to pyrimethamine
[1,49]. SP plus artesunate combination therapy has been
investigated in small trials as treatment for both
P. falciparum and P. vivax in Papua New Guinea,
including patients with mixed infection, suggesting that
SP –artesunate clears parasites of both species rapidly, but
that increases in the frequency of mutations in the
dihydrofolate reductase (dhfr) gene threaten its efficacy.
More research is required before this combination can be
recommended for mixed infections [49].
All other primary treatments for falciparum malaria kill
the asexual stages of all other human Plasmodium spp.
Where multidrug resistant P. falciparum exists, a combi-
nation of artesunate (4 mg kg21 day21) for three days) plus
mefloquine (15 mg kg21 on Day 1 followed by 10 mg kg21 on
Day 2), or artemether plus lumefantrine are both very
effective regimes [1]. However, these combinations will not
kill hypnozoites of P. vivax and P. ovale. Treatment of
patients with P. falciparum and P. vivax or P. ovale co-
infection should include a hypnozoitocidal drug (e.g.
primaquine 0.25–0.33 mg kg21 day21 for 14 days), for
radical cure of P. vivax and P. ovale. For mixed infections
with benign malaria species (P. vivax, P. ovale and
P. malariae), treatment with oral CQ (a total dose of 25 mg
over three days) followed by primaquine should be given.
However, CQ might not be effective in the areas where CQ-
resistant P. vivax exists, such as Papua New Guinea and
Indonesia [33], and the prevention of relapse during
pregnancy is not currently possible because primaquine is
contraindicated. Species-specific malaria vaccination and
control measures might also have an impact on the nature of
mixed-species infections. That this might occur is suggested
by the large increase in P. malariae prevalence associated
with a malaria control programme in Tanzania, which
reduced P. falciparum prevalence [18]. Indeed, in a trial of
the Spf66 vaccine in Brazil, an increased incidence of P. vivax
infections was found among those vaccinated, without a
statistically significant effect on the incidence of P. falci-
parum [50]. Such potentially important interactions will
need to be addressed in future vaccine trials.
Conclusions
Mixed malaria species infections are often underestimated.
PCR of admission blood samples and longitudinal studies of
patients, who have not been re-exposed to malaria after the
initial malaria attack, have demonstrated that mixed
infections are remarkably common, with the delayed
appearance of one cryptic species following treatment for
the species which was patent at presentation. Concurrently
infecting malaria species are mutually suppressive, with
P. falciparum tending to dominate P. vivax, but P. vivax
attenuating the severity of P. falciparum. There is little
evidence to guide the treatment of mixed infections, but the
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Vol.20 No.5 May 2004 239
available information suggests that SP and possibly
clindamycin are not appropriate for treating mixed
P. falciparum and P. vivax infections. Malaria control
measures and vaccination targeted at one malaria species
could affect the clinical epidemiology of sympatric Plas-
modium spp. In particular, widespread use of CQ could
worsen the severity of resistant falciparum malaria by
suppressing the other competing species.
Acknowledgements
We are grateful for very useful comments by anonymous reviewers and for
the help of Francois Nosten and Vasee Morthy. We apologize to all those
whose work we have been unable to discuss and cite due to space
constraints. The authors are supported by the Wellcome Trust of the UK.
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