2008 co infecções de p. falciparum


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2008 co infecções de p. falciparum

  1. 1. Acta Tropica 107 (2008) 8–12 Contents lists available at ScienceDirect Acta Tropica journal homepage: www.elsevier.com/locate/actatropicaMixed Plasmodium falciparum infections and its clinical implications infour areas of the Brazilian Amazon regionAlexandre Lorenzetti a , Patr´cia Aparecida Fornazari a , Ana Carolina Bonini-Domingos a , ı ´Roberta de Souza Rodrigues Penhalbel a , Erika Fugikaha a , Claudia Regina Bonini-Domingos b , ´Valeria Daltibari Fraga a , Luciana Moran Conceicao a , Andrea Regina Baptista Rossit a,d , ´ ¸˜ ´Carlos Eugˆ nio Cavasini a , Vanja Suely Calvosa D’Almeida Couto c , Ricardo Luiz Dantas Machado a,d,∗ ea Centro de Investigac˜o de Microrganismos, Departamento de Doencas Dermatol´gicas, Infecciosas e Parasit´rias, Faculdade de Medicina de S˜o Jos´ do Rio Preto, ¸a ¸ o a a eAvenida Brigadeiro Faria Lima 5416, 15090-000 S˜o Jos´ do Rio Preto, S˜o Paulo, Brazil a e ab Departamento de Biologia, Universidade de S˜o Paulo, Rua Crist´v˜o Colombo 2265, 15054-000 S˜o Jos´ do Rio Preto, S˜o Paulo, Brazil a o a a e ac Programa de Mal´ria, Instituto Evandro Chagas, Secretaria de Vigilˆncia em Sa´ de, BR316 Km 7, 67030-000 Ananindeua, Par´, Brazil a a u ad Fundac˜o Faculdade de Medicina de S˜o Jos´ do Rio Preto, Avenida Brigadeiro Faria Lima 5544, 15090-000 S˜o Jos´ do Rio Preto, S˜o Paulo, Brazil ¸a a e a e aa r t i c l e i n f o a b s t r a c tArticle history: The aim of this study was to assess the prevalence pattern of mixed-Plasmodium falciparum malaria infec-Received 16 July 2007 tions in Brazil by molecular diagnosis and to address its clinically important features. DNA was extractedReceived in revised form 26 February 2008 from 115 thick blood film P. falciparum human blood positive samples using the phenol–chloroformAccepted 27 March 2008 method, followed by a semi-nested PCR protocol with species-specific primers. Seventy-three percentAvailable online 4 April 2008 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 overKeywords: the other can be important on weakening P. falciparum malaria clinical symptoms. We confirm that P.Plasmodium falciparumMixed infection falciparum co-infections frequently occur in Brazilian malaria endemic areas, with underestimated diag-Clinical aspects nosis. The results point to the need of improving microscopy or changing for another accurate diagnosisBrazilian Amazon region 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 (1988) reported that mixed infections occurred less than would be expected from cross-sectional prevalences of the individual species. Malaria is an infectious disease caused by four species of Molineaux et al. (1980) reviewed prevalence surveys and concludedintraerythrocytic protozoan parasite of the Plasmodium genus. Of that mixed-species infections were actually more common thanthe known human malaria parasites, only Plasmodium falciparum, expected and Richie (1988) showed no general pattern in their fre-Plasmodium vivax, and Plasmodium malariae have been detected in quencies. In contrast, other studies found that lower than expected ´Brazil with 99.7% of cases occurring in the Amazon region (Povoa frequencies of dual P. vivax–P. falciparum infections correspond to ´ ´et al., 2000; Ministerio da Saude, 2003). a higher overall malaria prevalence and also that P. malariae–P. fal- Previous reports suggest that in mixed infections, one Plasmod- ciparum infections are invariably correlated with a large numberium species may suppress the blood-stage density of another (Hill of mixed infections (McKenzie and Bossert, 1997, 1999). Addition-et al., 1943; Bruce-Chwatt, 1963). Over the last years, the rela- ally, a mathematical model of the P. malariae–P. falciparum infectiontionships among the human malaria parasite infections have been versus the human host dynamics suggested that a P. malariae infec-investigated with contradictory results. Cohen’s (1973) and Richie 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 ∗ Corresponding author at: Centro de Investigacao de Microrganismos, Departa- ¸˜ more common in P. falciparum single infected patients than in ´ ´mento de Doencas Dermatologicas, Infecciosas e Parasitarias, Faculdade de Medicina ¸ those with dual P. falciparum–P. vivax infections (Luxemberger et ´ ´de S˜ o Jose do Rio Preto, Avenida Brigadeiro Faria Lima 5416, 15090-000 S˜ o Jose do a aRio Preto, S˜ o Paulo, Brazil. Tel.: +55 17 3201 5736; fax: +55 17 3201 5909. a al., 1997). Black et al. (1994) suggested that P. malariae infections E-mail address: ricardomachado@famerp.br (R.L.D. Machado). reduce the severity of subsequent P. falciparum infections, and that0001-706X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.actatropica.2008.03.012
  2. 2. A. Lorenzetti et al. / Acta Tropica 107 (2008) 8–12 9individuals concurrently infected by both species have experienced symptoms were defined as “present” or “absent” by the medicalsignificant reductions in fever. Recently, it was reported in Thailand staff accordingly to the temperature measurements performed bythat patients with dual P. vivax–P. falciparum infections have higher the nurses and also by a detailed, specific interview, regardingfevers than those with single-species infections (McKenzie et al., unusual and/or previously experienced clinical malaria manifes-2006). tation. Previous studies have pointed to highly relevant limitations oftraditional microscopy-based detection techniques (Snounou et al., 2.3. Laboratory analysis1993; Postigo et al., 1998). Indeed, the deficiency to detect mixedinfections by the thin and thick blood film methods make treatment Thick blood films (TBFs) were confirmed by independent expe-difficult as it is species-specific. Polymerase chain reaction (PCR) rienced microscopists who were unaware of each result accordinghas been shown to be efficient in the diagnosis of the four human to the World Health Organization recommended procedures. Bloodmalaria parasites and, therefore, also on identifying high prevalence samples were stored at −20 ◦ C until laboratory analyses. Samplesof mixed infections (Roper et al., 1996; May et al., 1999). The aim were treated with Proteinase K, and nucleic acids were extracted byof this study was to assess the prevalence pattern of mixed-P. fal- using two rounds of phenol:chloroform:isoamyl alcohol (25:24:1),ciparum malaria infections in Brazil by molecular diagnosis and to one round of chloroform and one of ether, followed by ethanol pre-address its clinically important features. cipitation. The extracted nucleic acid samples were dissolved in sterile pure deionised water, and stored at −20 ◦ C prior to use. The2. Materials and methods semi-nested PCR was based on the protocol accordingly to Kimura et al. (1997). The target was the SSU rDNA gene, and species-specific2.1. Study population primers were used in the assay. Briefly, the first PCR rDNA ampli- fication was performed with Plasmodium genus-specific primers. Sample collection took place from May 2003 to August 2005. Positive samples served as template for the nested reaction. TheOne hundred and fifteen male and female malaria patients nested PCR amplifications were performed using P. falciparum, P. ´from four regions of the Brazilian Amazon: Macapa, state of vivax, and P. malariae SSU rDNA primers plus universal primer fromAmapa (00◦ 02 20 S; 51◦ 03 59 W); Novo Repartimento, state of ´ the first reaction. The fragments obtained were seen at about 110-Para (04◦ 19 50 S; 49◦ 47 47 W), Porto Velho, state of Rondˆ nia ´ o bp. As a positive control we used blood samples with P. falciparum,(−08◦ 45 43 S; 63◦ 54 14 W); and Placido de Castro, state of Acre ´ P. vivax, and P. malariae TBF plus molecular results to Plasmodium.(10◦ 16 33 S; 67◦ 09 00 W) were enrolled in this study. These indi- As a negative control we used blood samples from blood donorsviduals presented on their own initiative, and were invited to living in the same areas with negative microscopy and molecularparticipate in this study at the public healthcare clinics in each results to Plasmodium. The products were visualized in 2% agarosestudy area. They were all over the age of 18 and had positive gel stained with ethidium bromide.thick blood film (TBF) results for P. falciparum single infection. Weexcluded from the study pregnant women, patients under the age of 2.4. Data analysis18 years and no other concomitant illness. Participants were askedto sign a written consent form before blood samples were drawn. Epi Info version 6.04b (CDC, Atlanta, US) was used for data stor-The consent form was co-signed by a staff member of the clinic. age and statistical analyses. Proportions and categorical data wereClinical and epidemiological data such as age, gender, past history compared by the Chi-square test, with Yate’s correction, in casesof malaria, and current infection information were obtained from a of 2 × 2 contingency tables, or Fisher exact test (two-tailed). Thespecific interview conducted by the physicians and also from med- adopted significance level for statistical inference was p < 0.05.ical records. The protocol for this study was reviewed and approved a ´by the Research Board of the Faculty of Medicine from S˜ o Jose do 3. ResultsRio Preto. The parasitaemia on the thick blood films ranged from 25 to2.2. Clinical evaluation 6500 parasites/mm3 . P. falciparum parasitaemia was lower among patients with mixed infections than among patients with single- All patients voluntarily sought medical assistance presenting species infections, but this difference was insignificant (Chi-squarewith uncomplicated clinical malaria symptoms as evaluated by ´ 5403, p > 0.7137). In Macapa patients, the previous malaria experi-the physicians and/or nurses enrolled in the malaria diagnosis and ence (in number of episodes) was 1.5 (±2.01); in those from Portotreatment routine of the Brazilian government national program. Velho was 0.9 (±1.57); in those from Novo Repartimento was 1.7Individuals who presented at least one of the following symptoms: ´ (±2.62) and from Placido de Castro was 1.6 (±2.57). As for theirfever, headache, and shiver, in addition to microscopic positivity, ages, the geometric means in each area were 28 (±1.35), 25 (±2.35),were included in the post-diagnostic medical evaluation. Likewise, 32 (±1.15), and 30 (±1.02) years old, respectively, ranging from 18Table 1Identification 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. malariae P. falciparum + P. vivax P. falciparum + P. malariae + P. vivaxNovo Repartimento/PA (n = 16) 14 (16.67%) – 2 (7.14%) – ´Macapa/AP (n = 37) 26 (30.95%) 1 (50%) 10 (35.71%) –Porto Velho/RO (n = 50) 35 (41.67%) – 14 (50%) 1 (100%) ´Placido de Castro/AC (n = 12) 9 (10.71%) 1 (50%) 2 (7.14%) –Total 84 2 28 1 ´ ´PA: Para; AP: Amapa; RO: Rondˆ nia; AC: Acre. o
  3. 3. 10 A. Lorenzetti et al. / Acta Tropica 107 (2008) 8–12Table 2 New Guinea (Mehlotra et al., 2000), and almost equally often in theFrequency (%) of clinical aspects (fever, headache and shiver) as function of P. falci- subjects from Guinea Bissau (Snounou et al., 1993), Laos (Toma etparum malaria attacks from Brazilian Amazon region, May 2003 to August 2005 al., 2001), and Mozambique (Marques et al., 2005). Several reportsClinical aspects P. falciparum infections demonstrate that P. falciparum infections may be influenced by the Single (n = 84) Mixed (n = 31) p presence of a congener (Mason et al., 1999) and frequently sup-Fever 84 (100%) 28 (90.32%) 0.0182 press P. vivax in cases of co-inoculation (Boyd and Kitchen, 1937;Headache 75 (89.29%) 23 (75.19%) 0.0461 Garham et al., 1956; Looareesuwan et al., 1987). These data con-Shiver 78 (92.86%) 24 (77.42%) 0.0405 firm that P. falciparum co-infections frequently occur in Brazilianp values are based on Fisher exact test. 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 formsto 52 years in all studied areas. As summarized in Table 1, 73.04% dynamics.of P. falciparum single infections and 26.95% of mixed infections When mixed-infection is misdiagnosed as a P. vivax single-were found. Amongst mixed infections, the majority was double species infection, treatment can lead to a surge in P. falciparuminfection (96.77%). parasitaemia (Mason and McKenzie, 1999). Many factors confound Of all the clinical aspects recorded during the 115 P. falciparum the relationship between parasitaemia and disease, but there ismalaria attacks, a typical febrile paroxysm was the most frequent generally a loose positive correlation between circulating para-clinical symptom, observed in 97.39% of cases, as a single or an site load and clinical status. Conversely, previous reports suggestassociated manifestation. The combination among the three clini- that P. vivax–P. falciparum interactions in mixed infections maycal aspects assessed (fever, headache and shiver) showed fever plus have profound clinical effects in uncomplicated malaria, perhapsheadache in 85.21% of cases, while fever plus shiver was reported by maintaining P. falciparum densities below the fever thresholdin 88.69%. There was a lower frequency of individuals presenting (Field, 1949; White, 1997). An explanation for the reduction of theclinical manifestations in the P. falciparum mixed-infections group symptom in mixed infection carriers could be the mean age of thecompared to the P. falciparum single infections one (Fisher exact affected patients and time of residence in the endemic area, sincetest, p < 0.05; Table 2). Clinical aspects were not correlated with it is well documented by different authors that immunity can playtotal parasitaemia (Chi-square 0.930, p > 0.99). There is no correla- an important role in malaria symptom relief (Alves et al., 2002;tion between the individuals’ age or past history of malaria and the Coura et al., 2006). Other possibility could be related to the num-reduction of their symptom in all study areas. ber of previous malaria episodes (Coura et al., 2006), but in the present investigation we were not able to find a positive correlation4. Discussion in all the raised points. In our study, the mean number of previous malaria episodes was low and the majority of the patients are living Although P. vivax is the most common human malaria parasite under 5 years in the endemic areas. In fact, in the Brazilian Amazonin Brazil, P. falciparum accounts for approximately 30% of overall region P. vivax and P. falciparum malaria predominate in Mesoen-cases, and is a greater cause of morbidity and mortality. The dis- demic conditions with wide variations in transmission, as it can betribution of P. falciparum infection is focal, more common than P. observed by the non-immune or semi-immune status of the adultvivax in some areas, but very rare or absent in others (Camargo population as well as by the asymptomatic carriers (Alves et al.,et al., 1999). Genetic divergence between Brazilian P. falciparum 2002; Coura et al., 2006). Consequently, minor clinical malaria evi-populations is very substantial with distinct population structures dence was referred by the studied patients once a reduction in theand minimal gene flow and these aspects may affect the rate of severity of malaria symptoms was reported in individuals with lim-increasing drug resistance. This is consistent with the view that P. ited pre-exposure to different species (Gunewardena et al., 1994).falciparum malaria in the largest endemic region of the Americas Another possible reason for the lack of association with age and pastshould not be seen as a single entity, and different strategies for history of malaria relies on McKenzie et al. (2006) findings, suggest-prevention and control may be designed for its diverse endemic ing that parasitaemia is not the most important symptom trigger.locations (Machado et al., 2004). On the other hand, in mixed- They indicate that one species prevalence over the other can bephenotype (drug resistant and sensitive) P. falciparum infection, important since individuals with P. vivax higher parasitaemia overineffective treatment can lead to higher densities of the resistant P. falciparum show fever reduction compared to those with higherprotozoan (Mason and McKenzie, 1999). It may be necessary to P. falciparum number of parasites. Concurrently, infecting malariaassess the prevalence of genotypes and/or mixed-species infections species are equally suppressive with P. falciparum dominating P.before control measures are implemented (Marques et al., 2005). vivax, but P. vivax attenuating the clinical complications of P. falci- Mixed infections diagnosed by microscopy in patients admit- parum (Mayxay et al., 2004). This last affirmative can explain theted in healthcare malaria clinics and in epidemiological surveys clinical observations occurring in our studied patients. On the otherare a small proportion of the total prevalence (McKenzie and hand, it is not a general consensus that higher fevers, per se, areBossert, 1999), but almost all combinations of species have been consequences of greater clinical severity or more effective immunefound within human populations and individuals (Mckenzie et al., responses (McKenzie et al., 2006).2002). Interestingly, Bruce et al. (2000) explore species interac- In spite of the fact that we do not have data on exact oral temper-tions through the interplay between density dependent regulation ature measurements and the subjective character of patient’s reportand differential growth and clearance rates of individual parasite on headache and shiver, a limitation we acknowledge, we observedpopulations. Growth of one parasite population to above threshold that P. falciparum mixed infections are associated with reduc-density would trigger density-dependent regulation thus inhibiting tion in the prevalence of these three symptoms in this samplingminority co-infections. P. falciparum mixed-infections, in this study, of the Brazilian Amazon region. Nevertheless, symptom reduc-were identified in 26.95% of all samples studied. In previous molec- tion was not correlated with total parasitaemia. Finally, since theular studies from Brazil, P. falciparum mixed-species were detected virulence has been shown to be associated with selectivity ofin 23.96% (Cavasini et al., 2000), 20% (Alves et al., 2002), and 17.62% erythrocyte invasion (Chotivanich et al., 2000), an overall under-(Scopel et al., 2004). All these frequencies were lower than those standing of the biological interactions of the parasite/host is suchreported in studies from Thailand (Zhou et al., 1998) and Papua that one could be imperative in clinical implications (McKenzie et
  4. 4. A. Lorenzetti et al. / Acta Tropica 107 (2008) 8–12 11al., 2006). For instance, we could observe dramatic differences in ´ Jose do Rio Preto. Financial support: FAPESP (02/09546-1) and CNPqP. falciparum Brazilian population’s structure compared to Thailand (302353/03-8).(Anderson et al., 2000), since heterozygosity in Brazilian parasitesis lower (Machado et al., 2004). Furthermore, innate resistance Referencesto malaria infections in humans was attributed to blood groupantigen variations. Invasion of red blood cells (RBC) occurs when Alves, F.P., Durlacher, R.R., Menezes, M.J., Krieger, H., Silva, L.H.P., Camargo, E.P., 2002.the extracellular form of the parasite, the merozoites, attaches to High prevalence of asymptomatic Plasmodium vivax and Plasmodium falciparumthe surface of an uninfected RBC. We observed significant cor- infections in native Amazonian populations. Am. J. Trop. Med. Hyg. 66, 641–648. Anderson, T.J.C., Haubold, B., Williams, J.T., Estrada-Franco, J.G., Richardson, L.,relation regarding MNSs (P. falciparum erythrocyte receptor) and Mollinedo, R., Bockarie, M., Mokili, J., Mharakurwa, S., French, N., Whitworth, J.,Duffy blood (acts as a RBC receptor for P. vivax) group pheno- Velez, I.D., Brockman, A., Nosten, F., Ferreira, M.U., Day, K.P., 2000. Microsatellitestypes between blood donors and malaria patients in the same reveal a spectrum of population structures in the malaria parasite Plasmodium falciparum. Mol. Biol. Evol. 17, 1467–1482.studied areas (Cavasini et al., 2006). The results of the current Black, J., Hommel, M., Snounou, G., Pinder, M., 1994. Mixed infections with Plasmod-work suggest that P. falciparum mixed-infections can affect Brazil- ium vivax and Plasmodium malariae and fever in malaria. Lancet 343, 1095.ian transmission patterns by attenuation of P. falciparum disease Boyd, M.F., Kitchen, S.F., 1937. Simultaneous inoculation with Plasmodium vivax and(Black et al., 1994) and ultimately may have an effect in the way this Plasmodium falciparum. Am. J. Trop. Med. Hyg. 17, 855–861. Bruce-Chwatt, L.J., 1963. A longitudinal survey of natural infection in a group of Westspecies establishes itself in the population. Further studies based African adults. Part I. West African Med. J. 12, 141–143.on accurate oral fever measurement accompanied by other symp- Bruce, M.C., Donnelly, C.A., Alpers, M.P., Galinski, M.R., Barnwell, J.W., Walliker, D.,toms evaluation, in different human populations and localities, will Day, K.P., 2000. Cross-species interactions between malaria parasites in humans. Science 287, 845–848.clarify the importance of Plasmodium mixed infections in malaria Camargo, L.M.A., Noronha, E., Salcedo, V.J., Dutra, A.P., Kriger, H., Pereira da Silva, L.H.,clinical outcome. Camargo, E.P., 1999. The epidemiology of malaria in Rondˆ nia (Western Amazon o The apparent frequency of mixed infections is dependent on region, Brazil): study of a riverine population. Acta Trop. 72, 1–11. Cavasini, C.E., Mattos, L.C., Alves, R.T., Couto, A.A., Calvosa, V.S., Domingos, C.R.,the technique used for parasite analyses. An inadequate P. falci- Castilho, L., Rossit, A.R., Machado, R.L., 2006. Frequencies of ABO, MNSs, andparum diagnosis within a mixed infection could result in incorrect Duffy phenotypes among blood donors and malaria patients from four Braziliantreatment and consequently, in a severer disease (Mayxay et al., Amazon areas. Hum. Biol. 78, 215–219. Cavasini, M.T.V., Ribeiro, W.L., Kawamoto, F., Ferreira, U.M., 2000. How prevalent is2004). Double-species infections can commonly be detected by Plasmodium malariae in Rondˆ nia, Western Brazilian Amazon? Rev. Soc. Bras. oboth microscopy and PCR methods; whereas most triple-species Med. Trop. 33, 489–492.infections are frequently detected only by molecular techniques Chotivanich, K., Udomsangpetch, R., Simpson, J.A., Newton, P., Pukrittayakamee, S., Looareeswan, S., White, N.J., 2000. Parasite multiplication potential and the(May et al., 2000). Our results confirm that Plasmodium mixed infec- severity of falciparum malaria. J. Infect. Dis. 181, 1206–1209.tions diagnosis is underestimated in the four studied areas. Some Cohen, J.E., 1973. Heterologus immunity in human malaria. Quart. Rev. Biol. 48,limitations of thick blood film could result in misdiagnose, such 467–489.as the difficulty in distinguishing the young-form parasites of the Coura, J.R., Suarez-Mutis, M., Ladeia-Andrade, S., 2006. A new challenge for malaria control in Brazil: asymptomatic Plasmodium infection—a review. Mem. 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Oswaldo Cruz 89, 63–65.of Health in Brazil regarding the TBF control after treatment of the Hill, R.B., Cambournac, F.J.C., Sim˜ es, M.P., 1943. Observations on the course of oinitial malaria attack, performed 3–4 weeks following initial diag- malaria in children in an endemic region. Am. J. Trop. Med. Hyg. 23, 147–nosis, one can still figure the problem of up to a month of a second 162. Kimura, M., Kneko, O., Liu, Q., Zhou, M., Kawamoto, F., Wataya, Y., Otani, S., Yam-Plasmodium non-eliminated species carrier within the local popula- aguchi, Y., Tanake, K., 1997. Identification of the four species of human malariation. Even so, considering the Brazilian health system conditions, it parasites by nested PCR that targets variant sequences in the small subunit rRNAis impossible to include species-specific PCR as a routine in malaria gene. Parasitol. Intern. 46, 91–95. Looareesuwan, S., White, N.J., Chittamas, S., Bunnag, D., Harinasuta, T., 1987. Highdiagnosis. Therefore, we propose that a plausible action would be rate of Plasmodium vivax relapse following treatment of falciparum malaria into improve TBF conditions in the field by simple proceedings like Thailand. Lancet 2, 1052–1055.equipment maintenance and technician’s supervision and support. Luxemberger, C., Ricci, F., Nosten, F., Raimond, D., Bathet, S., White, N.J., 1997. The In conclusion, our results point to the need of improving epidemiology of severe malaria in an area of low transmission in Thailand. Trans. R. Soc. Trop. Med. Hyg. 91, 256–262.microscopy or changing for another accurate diagnosis method, ´ Machado, R.L.D., Povoa, M.M., Calvosa, V.S.P., Ferreira, M.U., Rossit, A.R.B., Santos,especially in endemic areas, to differentiate among human malaria E.J.M., Conway, D.C., 2004. Genetic structure of Plasmodium falciparum popula-species. If some interactions between species are such that one tions in the Brazilian Amazon region. J. Infect. Dis. 190, 1547–1555. ´ ´ Marques, P.X., Saute, F., Pinto, V.V., Cardoso, S., Pinto, J., Alonso, P.L., Rosario, V.E.,may affect by even minor differences the clinical manifestation of Arez, A.P., 2005. Plasmodium species mixed infections in two areas of Manhica ¸another, more investigations are necessary in order to clarify the District, Mozambique. Int. J. Biol. Sci. 1, 96–102.role of mixed-infections in P. falciparum disease severity and also Mason, D.P., McKenzie, F.E., 1999. Blood-stage dynamics and clinical implications of mixed Plasmodium vivax–Plasmodium falciparum infections. Am. J. Trop. Med.in this parasite transmission dynamics. Hyg. 61, 367–374. Mason, D.P., McKenzie, F.E., Bossert, W.H., 1999. The blood-stage dynamics of mixed Plasmodium malariae–Plasmodium falciparum infections. J. Theor. Biol. 198, 549–566.Acknowledgements May, J., Falusi, A.G., Mockenhaupt, F.P., Ademowo, O.G., Olumese, P.E., Bienzle, U., Meyer, C.G., 2000. Impact of subpatent multi-species and multi-clonal plas- To the population enrolled in this study. To Aline Barroso, Maria modial infections on anaemia in children from Nigeria. Trans. R. Soc. Trop. Med. Hyg. 94, 399–403.Cristina Figueredo and Mauro Tada for help in field work. To Pro- May, J., Mokenhaupt, F.P., Ademowo, O.G., Olumese, P.E., Bienzle, U., Meyer, C.G., 1999.fessor Luiz Hildebrando Pereira da Silva for facilities at Cepem. High rate of mixed and subpatent malarial infections in southwest Nigeria. J. ´To Dr. Marinete Marins Povoa and Alexandre Moura for the com- Infect. Dis. 61, 339–343.ments and suggestions. P.A.F. is research studentship from Fapesp Mayxay, M., Khanthavong, M., Lindegardh, N., Keola, S., Barends, M., Pongvongsa, T., Yapom, R., Annerberg, A., Phompida, S., Phetsouvanh, R., White, N.J., Newton, P.N.,and A.L., A.C.B.D. and R.S.R.P. from CNPq. A.R.B.R. and R.L.D.M. are 2004. Randomized comparison of chloroquine plus sulfadoxine-pyrimethamine ¸˜research fellowship from Fundacao Faculdade de Medicina de S˜ o a versus artesunate plus mefloquine versus artemether-lumefantrine in the treat-
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