Mixed Plasmodium falciparum infections and its clinical implications in four areas of the Brazilian Amazon region.

Abstract

The aim of this study was to assess the prevalence pattern of mixed-Plasmodium falciparum malaria infections in Brazil by molecular diagnosis and to address its clinically important features. DNA was extracted from 115 thick blood film P. falciparum human blood positive samples using the phenol-chloroform method, followed by a semi-nested PCR protocol with species-specific primers. Seventy-three percent of P. falciparum single infections and 26.95% of mixed infections were found. Amongst mixed infections, the majority was double infection (96.77%). Our results suggest that the prevalence of one species over the other can be important on weakening P. falciparum malaria clinical symptoms. We confirm that P. falciparum co-infections frequently occur in Brazilian malaria endemic areas, with underestimated diagnosis. The results point to the need of improving microscopy or changing for another accurate diagnosis technique to differentiate among human malaria species, as this is essential to choose the best treatment and control measure for malaria. More investigations are necessary in order to clarify the role of mixed-infections in the severity of P. falciparum disease.

Full text

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Acta Tropica 107 (2008) 8–12
Contents lists available at ScienceDirect
Acta Tropica
journal homepage: www.elsevier.com/locate/actatropica
Mixed Plasmodium falciparum infections and its clinical implications in
four areas of the Brazilian Amazon region
Alexandre Lorenzettia, Patr´ ıcia Aparecida Fornazaria, Ana Carolina Bonini-Domingosa,
Roberta de Souza Rodrigues Penhalbela,´Erika Fugikahaa, Cl´ audia Regina Bonini-Domingosb,
Val´ eria Daltibari Fragaa, Luciana Moran Conceic ¸ ˜ aoa, Andr´ ea Regina Baptista Rossita,d,
Carlos Eugˆ enio Cavasinia, Vanja Suely Calvosa D’Almeida Coutoc, Ricardo Luiz Dantas Machadoa,d,∗
aCentro de Investigac ¸˜ ao de Microrganismos, Departamento de Doenc ¸as Dermatol´ ogicas, Infecciosas e Parasit´ arias, Faculdade de Medicina de S˜ ao Jos´ e do Rio Preto,
Avenida Brigadeiro Faria Lima 5416, 15090-000 S˜ ao Jos´ e do Rio Preto, S˜ ao Paulo, Brazil
bDepartamento de Biologia, Universidade de S˜ ao Paulo, Rua Crist´ ov˜ ao Colombo 2265, 15054-000 S˜ ao Jos´ e do Rio Preto, S˜ ao Paulo, Brazil
cPrograma de Mal´ aria, Instituto Evandro Chagas, Secretaria de Vigilˆ ancia em Sa´ ude, BR316 Km 7, 67030-000 Ananindeua, Par´ a, Brazil
dFundac ¸˜ ao Faculdade de Medicina de S˜ ao Jos´ e do Rio Preto, Avenida Brigadeiro Faria Lima 5544, 15090-000 S˜ ao Jos´ e do Rio Preto, S˜ ao Paulo, Brazil
a r t i c l e i n f o
Article history:
Received 16 July 2007
Received in revised form 26 February 2008
Accepted 27 March 2008
Available online 4 April 2008
Keywords:
Plasmodium falciparum
Mixed infection
Clinical aspects
Brazilian Amazon region
a b s t r a c t
The aim of this study was to assess the prevalence pattern of mixed-Plasmodium falciparum malaria infec-
tions in Brazil by molecular diagnosis and to address its clinically important features. DNA was extracted
from 115 thick blood film P. falciparum human blood positive samples using the phenol–chloroform
method, followed by a semi-nested PCR protocol with species-specific primers. Seventy-three percent
of P. falciparum single infections and 26.95% of mixed infections were found. Amongst mixed infections,
the majority was double infection (96.77%). Our results suggest that the prevalence of one species over
the other can be important on weakening P. falciparum malaria clinical symptoms. We confirm that P.
falciparum co-infections frequently occur in Brazilian malaria endemic areas, with underestimated diag-
nosis. The results point to the need of improving microscopy or changing for another accurate diagnosis
technique to differentiate among human malaria species, as this is essential to choose the best treat-
ment and control measure for malaria. More investigations are necessary in order to clarify the role of
mixed-infections in the severity of P. falciparum disease.
© 2008 Elsevier B.V. All rights reserved.
1. Introduction
Malaria is an infectious disease caused by four species of
intraerythrocytic protozoan parasite of the Plasmodium genus. Of
the known human malaria parasites, only Plasmodium falciparum,
Plasmodium vivax, and Plasmodium malariae have been detected in
Brazil with 99.7% of cases occurring in the Amazon region (P´ ovoa
et al., 2000; Minist´ erio da Sa´ ude, 2003).
Previous reports suggest that in mixed infections, one Plasmod-
ium species may suppress the blood-stage density of another (Hill
et al., 1943; Bruce-Chwatt, 1963). Over the last years, the rela-
tionships among the human malaria parasite infections have been
investigated with contradictory results. Cohen’s (1973) and Richie
∗Corresponding author at: Centro de Investigac ¸ ˜ ao de Microrganismos, Departa-
mentodeDoenc ¸asDermatol´ ogicas,InfecciosaseParasit´ arias,FaculdadedeMedicina
de S˜ ao Jos´ e do Rio Preto, Avenida Brigadeiro Faria Lima 5416, 15090-000 S˜ ao Jos´ e do
Rio Preto, S˜ ao Paulo, Brazil. Tel.: +55 17 3201 5736; fax: +55 17 3201 5909.
E-mail address: ricardomachado@famerp.br (R.L.D. Machado).
(1988) reported that mixed infections occurred less than would be
expectedfromcross-sectionalprevalencesoftheindividualspecies.
Molineauxetal.(1980)reviewedprevalencesurveysandconcluded
that mixed-species infections were actually more common than
expected and Richie (1988) showed no general pattern in their fre-
quencies. In contrast, other studies found that lower than expected
frequencies of dual P. vivax–P. falciparum infections correspond to
a higher overall malaria prevalence and also that P. malariae–P. fal-
ciparum infections are invariably correlated with a large number
of mixed infections (McKenzie and Bossert, 1997, 1999). Addition-
ally,amathematicalmodeloftheP.malariae–P.falciparuminfection
versus the human host dynamics suggested that a P. malariae infec-
tion can reduce the peak parasitemia of a subsequent P. falciparum
infection (Mason et al., 1999).
Cohen’s (1973) reports associated a decreased spleen size with
mixed infections. Severe malaria was approximately four times
more common in P. falciparum single infected patients than in
those with dual P. falciparum–P. vivax infections (Luxemberger et
al., 1997). Black et al. (1994) suggested that P. malariae infections
reduce the severity of subsequent P. falciparum infections, and that
0001-706X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.actatropica.2008.03.012

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A. Lorenzetti et al. / Acta Tropica 107 (2008) 8–12
9
individualsconcurrentlyinfectedbybothspecieshaveexperienced
significantreductionsinfever.Recently,itwasreportedinThailand
that patients with dual P. vivax–P. falciparum infections have higher
fevers than those with single-species infections (McKenzie et al.,
2006).
Previous studies have pointed to highly relevant limitations of
traditionalmicroscopy-baseddetectiontechniques(Snounouetal.,
1993; Postigo et al., 1998). Indeed, the deficiency to detect mixed
infectionsbythethinandthickbloodfilmmethodsmaketreatment
difficult as it is species-specific. Polymerase chain reaction (PCR)
has been shown to be efficient in the diagnosis of the four human
malariaparasitesand,therefore,alsoonidentifyinghighprevalence
of mixed infections (Roper et al., 1996; May et al., 1999). The aim
of this study was to assess the prevalence pattern of mixed-P. fal-
ciparum malaria infections in Brazil by molecular diagnosis and to
address its clinically important features.
2. Materials and methods
2.1. Study population
Sample collection took place from May 2003 to August 2005.
One hundred and fifteen male and female malaria patients
from four regions of the Brazilian Amazon: Macap´ a, state of
Amap´ a (00◦02?20??S; 51◦03?59??W); Novo Repartimento, state of
Par´ a (04◦19?50??S; 49◦47?47??W), Porto Velho, state of Rondˆ onia
(−08◦45?43??S; 63◦54?14??W); and Pl´ acido de Castro, state of Acre
(10◦16?33??S; 67◦09?00??W) were enrolled in this study. These indi-
viduals presented on their own initiative, and were invited to
participate in this study at the public healthcare clinics in each
study area. They were all over the age of 18 and had positive
thick blood film (TBF) results for P. falciparum single infection. We
excludedfromthestudypregnantwomen,patientsundertheageof
18 years and no other concomitant illness. Participants were asked
to sign a written consent form before blood samples were drawn.
The consent form was co-signed by a staff member of the clinic.
Clinical and epidemiological data such as age, gender, past history
of malaria, and current infection information were obtained from a
specific interview conducted by the physicians and also from med-
icalrecords.Theprotocolforthisstudywasreviewedandapproved
by the Research Board of the Faculty of Medicine from S˜ ao Jos´ e do
Rio Preto.
2.2. Clinical evaluation
All patients voluntarily sought medical assistance presenting
with uncomplicated clinical malaria symptoms as evaluated by
the physicians and/or nurses enrolled in the malaria diagnosis and
treatment routine of the Brazilian government national program.
Individuals who presented at least one of the following symptoms:
fever, headache, and shiver, in addition to microscopic positivity,
were included in the post-diagnostic medical evaluation. Likewise,
symptoms were defined as “present” or “absent” by the medical
staff accordingly to the temperature measurements performed by
the nurses and also by a detailed, specific interview, regarding
unusual and/or previously experienced clinical malaria manifes-
tation.
2.3. Laboratory analysis
Thick blood films (TBFs) were confirmed by independent expe-
rienced microscopists who were unaware of each result according
totheWorldHealthOrganizationrecommendedprocedures.Blood
samples were stored at −20◦C until laboratory analyses. Samples
weretreatedwithProteinaseK,andnucleicacidswereextractedby
using two rounds of phenol:chloroform:isoamyl alcohol (25:24:1),
one round of chloroform and one of ether, followed by ethanol pre-
cipitation. The extracted nucleic acid samples were dissolved in
sterile pure deionised water, and stored at −20◦C prior to use. The
semi-nested PCR was based on the protocol accordingly to Kimura
etal.(1997).ThetargetwastheSSUrDNAgene,andspecies-specific
primers were used in the assay. Briefly, the first PCR rDNA ampli-
fication was performed with Plasmodium genus-specific primers.
Positive samples served as template for the nested reaction. The
nested PCR amplifications were performed using P. falciparum, P.
vivax, and P. malariae SSU rDNA primers plus universal primer from
the first reaction. The fragments obtained were seen at about 110-
bp. As a positive control we used blood samples with P. falciparum,
P. vivax, and P. malariae TBF plus molecular results to Plasmodium.
As a negative control we used blood samples from blood donors
living in the same areas with negative microscopy and molecular
results to Plasmodium. The products were visualized in 2% agarose
gel stained with ethidium bromide.
2.4. Data analysis
Epi Info version 6.04b (CDC, Atlanta, US) was used for data stor-
age and statistical analyses. Proportions and categorical data were
compared by the Chi-square test, with Yate’s correction, in cases
of 2×2 contingency tables, or Fisher exact test (two-tailed). The
adopted significance level for statistical inference was p<0.05.
3. Results
The parasitaemia on the thick blood films ranged from 25 to
6500parasites/mm3. P. falciparum parasitaemia was lower among
patients with mixed infections than among patients with single-
species infections, but this difference was insignificant (Chi-square
5403, p>0.7137). In Macap´ a patients, the previous malaria experi-
ence (in number of episodes) was 1.5 (±2.01); in those from Porto
Velho was 0.9 (±1.57); in those from Novo Repartimento was 1.7
(±2.62) and from Pl´ acido de Castro was 1.6 (±2.57). As for their
ages,thegeometricmeansineachareawere28(±1.35),25(±2.35),
32 (±1.15), and 30 (±1.02) years old, respectively, ranging from 18
Table 1
Identification of Plasmodium falciparum mixed-infections as determined by malaria genotypic test among 115 patients from four Brazilian Amazon areas
Molecular diagnosis
P. falciparum P. falciparum+P. malariaeP. falciparum+P. vivaxP. falciparum+P. malariae+P. vivax
Novo Repartimento/PA (n=16)
Macap´ a/AP (n=37)
Porto Velho/RO (n=50)
Pl´ acido de Castro/AC (n=12)
14(16.67%)
26(30.95%)
35(41.67%)
9(10.71%)
–
1 (50%)
–
1 (50%)
2(7.14%)
10(35.71%)
14 (50%)
2(7.14%)
–
–
1 (100%)
–
Total842281
PA: Par´ a; AP: Amap´ a; RO: Rondˆ onia; AC: Acre.

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A. Lorenzetti et al. / Acta Tropica 107 (2008) 8–12
Table 2
Frequency (%) of clinical aspects (fever, headache and shiver) as function of P. falci-
parum malaria attacks from Brazilian Amazon region, May 2003 to August 2005
Clinical aspects
P. falciparum infections
Single (n=84) Mixed (n=31)
p
Fever
Headache
Shiver
84 (100%)
75(89.29%)
78(92.86%)
28(90.32%)
23(75.19%)
24(77.42%)
0.0182
0.0461
0.0405
p values are based on Fisher exact test.
to 52 years in all studied areas. As summarized in Table 1, 73.04%
of P. falciparum single infections and 26.95% of mixed infections
were found. Amongst mixed infections, the majority was double
infection (96.77%).
Of all the clinical aspects recorded during the 115 P. falciparum
malaria attacks, a typical febrile paroxysm was the most frequent
clinical symptom, observed in 97.39% of cases, as a single or an
associated manifestation. The combination among the three clini-
calaspectsassessed(fever,headacheandshiver)showedfeverplus
headache in 85.21% of cases, while fever plus shiver was reported
in 88.69%. There was a lower frequency of individuals presenting
clinical manifestations in the P. falciparum mixed-infections group
compared to the P. falciparum single infections one (Fisher exact
test, p<0.05; Table 2). Clinical aspects were not correlated with
total parasitaemia (Chi-square 0.930, p>0.99). There is no correla-
tion between the individuals’ age or past history of malaria and the
reduction of their symptom in all study areas.
4. Discussion
Although P. vivax is the most common human malaria parasite
in Brazil, P. falciparum accounts for approximately 30% of overall
cases, and is a greater cause of morbidity and mortality. The dis-
tribution of P. falciparum infection is focal, more common than P.
vivax in some areas, but very rare or absent in others (Camargo
et al., 1999). Genetic divergence between Brazilian P. falciparum
populations is very substantial with distinct population structures
and minimal gene flow and these aspects may affect the rate of
increasing drug resistance. This is consistent with the view that P.
falciparum malaria in the largest endemic region of the Americas
should not be seen as a single entity, and different strategies for
prevention and control may be designed for its diverse endemic
locations (Machado et al., 2004). On the other hand, in mixed-
phenotype (drug resistant and sensitive) P. falciparum infection,
ineffective treatment can lead to higher densities of the resistant
protozoan (Mason and McKenzie, 1999). It may be necessary to
assesstheprevalenceofgenotypesand/ormixed-speciesinfections
before control measures are implemented (Marques et al., 2005).
Mixed infections diagnosed by microscopy in patients admit-
ted in healthcare malaria clinics and in epidemiological surveys
are a small proportion of the total prevalence (McKenzie and
Bossert, 1999), but almost all combinations of species have been
found within human populations and individuals (Mckenzie et al.,
2002). Interestingly, Bruce et al. (2000) explore species interac-
tions through the interplay between density dependent regulation
and differential growth and clearance rates of individual parasite
populations. Growth of one parasite population to above threshold
densitywouldtriggerdensity-dependentregulationthusinhibiting
minorityco-infections.P.falciparummixed-infections,inthisstudy,
were identified in 26.95% of all samples studied. In previous molec-
ular studies from Brazil, P. falciparum mixed-species were detected
in 23.96% (Cavasini et al., 2000), 20% (Alves et al., 2002), and 17.62%
(Scopel et al., 2004). All these frequencies were lower than those
reported in studies from Thailand (Zhou et al., 1998) and Papua
New Guinea (Mehlotra et al., 2000), and almost equally often in the
subjects from Guinea Bissau (Snounou et al., 1993), Laos (Toma et
al., 2001), and Mozambique (Marques et al., 2005). Several reports
demonstrate that P. falciparum infections may be influenced by the
presence of a congener (Mason et al., 1999) and frequently sup-
press P. vivax in cases of co-inoculation (Boyd and Kitchen, 1937;
Garham et al., 1956; Looareesuwan et al., 1987). These data con-
firm that P. falciparum co-infections frequently occur in Brazilian
malaria endemic areas and the pair P. falciparum–P. vivax seems to
be the commonest. This information needs further evaluation, in
order to measure infection and densities of asexual/sexual forms
dynamics.
When mixed-infection is misdiagnosed as a P. vivax single-
species infection, treatment can lead to a surge in P. falciparum
parasitaemia (Mason and McKenzie, 1999). Many factors confound
the relationship between parasitaemia and disease, but there is
generally a loose positive correlation between circulating para-
site load and clinical status. Conversely, previous reports suggest
that P. vivax–P. falciparum interactions in mixed infections may
have profound clinical effects in uncomplicated malaria, perhaps
by maintaining P. falciparum densities below the fever threshold
(Field, 1949; White, 1997). An explanation for the reduction of the
symptom in mixed infection carriers could be the mean age of the
affected patients and time of residence in the endemic area, since
it is well documented by different authors that immunity can play
an important role in malaria symptom relief (Alves et al., 2002;
Coura et al., 2006). Other possibility could be related to the num-
ber of previous malaria episodes (Coura et al., 2006), but in the
presentinvestigationwewerenotabletofindapositivecorrelation
in all the raised points. In our study, the mean number of previous
malariaepisodeswaslowandthemajorityofthepatientsareliving
under 5 years in the endemic areas. In fact, in the Brazilian Amazon
region P. vivax and P. falciparum malaria predominate in Mesoen-
demic conditions with wide variations in transmission, as it can be
observed by the non-immune or semi-immune status of the adult
population as well as by the asymptomatic carriers (Alves et al.,
2002; Coura et al., 2006). Consequently, minor clinical malaria evi-
dence was referred by the studied patients once a reduction in the
severityofmalariasymptomswasreportedinindividualswithlim-
ited pre-exposure to different species (Gunewardena et al., 1994).
Anotherpossiblereasonforthelackofassociationwithageandpast
historyofmalariareliesonMcKenzieetal.(2006)findings,suggest-
ing that parasitaemia is not the most important symptom trigger.
They indicate that one species prevalence over the other can be
important since individuals with P. vivax higher parasitaemia over
P. falciparum show fever reduction compared to those with higher
P. falciparum number of parasites. Concurrently, infecting malaria
species are equally suppressive with P. falciparum dominating P.
vivax, but P. vivax attenuating the clinical complications of P. falci-
parum (Mayxay et al., 2004). This last affirmative can explain the
clinicalobservationsoccurringinourstudiedpatients.Ontheother
hand, it is not a general consensus that higher fevers, per se, are
consequences of greater clinical severity or more effective immune
responses (McKenzie et al., 2006).
Inspiteofthefactthatwedonothavedataonexactoraltemper-
aturemeasurementsandthesubjectivecharacterofpatient’sreport
on headache and shiver, a limitation we acknowledge, we observed
that P. falciparum mixed infections are associated with reduc-
tion in the prevalence of these three symptoms in this sampling
of the Brazilian Amazon region. Nevertheless, symptom reduc-
tion was not correlated with total parasitaemia. Finally, since the
virulence has been shown to be associated with selectivity of
erythrocyte invasion (Chotivanich et al., 2000), an overall under-
standing of the biological interactions of the parasite/host is such
that one could be imperative in clinical implications (McKenzie et

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A. Lorenzetti et al. / Acta Tropica 107 (2008) 8–12
11
al., 2006). For instance, we could observe dramatic differences in
P. falciparum Brazilian population’s structure compared to Thailand
(Anderson et al., 2000), since heterozygosity in Brazilian parasites
is lower (Machado et al., 2004). Furthermore, innate resistance
to malaria infections in humans was attributed to blood group
antigen variations. Invasion of red blood cells (RBC) occurs when
the extracellular form of the parasite, the merozoites, attaches to
the surface of an uninfected RBC. We observed significant cor-
relation regarding MNSs (P. falciparum erythrocyte receptor) and
Duffy blood (acts as a RBC receptor for P. vivax) group pheno-
types between blood donors and malaria patients in the same
studied areas (Cavasini et al., 2006). The results of the current
work suggest that P. falciparum mixed-infections can affect Brazil-
ian transmission patterns by attenuation of P. falciparum disease
(Blacketal.,1994)andultimatelymayhaveaneffectinthewaythis
species establishes itself in the population. Further studies based
on accurate oral fever measurement accompanied by other symp-
tomsevaluation,indifferenthumanpopulationsandlocalities,will
clarify the importance of Plasmodium mixed infections in malaria
clinical outcome.
The apparent frequency of mixed infections is dependent on
the technique used for parasite analyses. An inadequate P. falci-
parum diagnosis within a mixed infection could result in incorrect
treatment and consequently, in a severer disease (Mayxay et al.,
2004). Double-species infections can commonly be detected by
both microscopy and PCR methods; whereas most triple-species
infections are frequently detected only by molecular techniques
(Mayetal.,2000).OurresultsconfirmthatPlasmodiummixedinfec-
tions diagnosis is underestimated in the four studied areas. Some
limitations of thick blood film could result in misdiagnose, such
as the difficulty in distinguishing the young-form parasites of the
human malaria species, the undetectable presence of P. vivax liver
hypnozoitesandthefactthatmanyinfectionsareatdensitiesbelow
thethresholddetectablebymicroscopy(Blacketal.,1994).Further-
more, microscopists, on finding one species, might not search the
slide for a rare second species (Zhou et al., 1998). In spite of the
recommendation by World Health Organization and the Ministry
of Health in Brazil regarding the TBF control after treatment of the
initial malaria attack, performed 3–4 weeks following initial diag-
nosis, one can still figure the problem of up to a month of a second
Plasmodiumnon-eliminatedspeciescarrierwithinthelocalpopula-
tion. Even so, considering the Brazilian health system conditions, it
is impossible to include species-specific PCR as a routine in malaria
diagnosis. Therefore, we propose that a plausible action would be
to improve TBF conditions in the field by simple proceedings like
equipment maintenance and technician’s supervision and support.
In conclusion, our results point to the need of improving
microscopy or changing for another accurate diagnosis method,
especially in endemic areas, to differentiate among human malaria
species. If some interactions between species are such that one
may affect by even minor differences the clinical manifestation of
another, more investigations are necessary in order to clarify the
role of mixed-infections in P. falciparum disease severity and also
in this parasite transmission dynamics.
Acknowledgements
To the population enrolled in this study. To Aline Barroso, Maria
Cristina Figueredo and Mauro Tada for help in field work. To Pro-
fessor Luiz Hildebrando Pereira da Silva for facilities at Cepem.
To Dr. Marinete Marins P´ ovoa and Alexandre Moura for the com-
ments and suggestions. P.A.F. is research studentship from Fapesp
and A.L., A.C.B.D. and R.S.R.P. from CNPq. A.R.B.R. and R.L.D.M. are
research fellowship from Fundac ¸ ˜ ao Faculdade de Medicina de S˜ ao
Jos´ edoRioPreto.Financialsupport:FAPESP(02/09546-1)andCNPq
(302353/03-8).
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