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
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
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. 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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. 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