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Am. J. Trop. Med. Hyg., 76(3), 2007, pp. 559–565 Copyright © 2007 by The American Society of Tropical Medicine and Hygiene


Abstract. Randomly amplified polymorphic DNA (RAPD) analysis was performed to infer the magnitude and pattern of genetic differentiation among Lutzomyia (Nyssomyia) intermedia populations from two ecologic regions in southeast Brazil where transmission of American cutaneous leishmaniasis (ACL) by L. (Viannia) braziliensis reflects distinct eco-epidemiologic features. Two hundred thirty-five bands from six primers were analyzed for genetic variation within and between sand fly populations. A lower level of intrapopulational variability was found in domestic sand flies (compared with the peridomestic population). Gene flow FST estimates indicated homogeneity in the studied population, with limited population substructuring, but with a tendency of sand fly vectors to form clusters by micro-habitat (domestic and peridomestic). The level of genetic structuring of Lu. intermedia population from each ecologic region is distinct and may reflect association or independence between the domestic and the peridomestic habitats in rural and periurban areas, respectively, both reflecting distinct characteristics of the transmission cycle of ACL. phenotypes14 of epidemiologic importance.15 Recently, we have studied gene flow between sylvatic and domestic populations of Lu. intermedia in a restricted focus of ACL (Mesquita, near Rio de Janeiro), where the genetic diversity of L. (V.) braziliensis is very low.9,16 Multi-locus DNA markers showed the existence of two major clusters associated with domiciliary and sylvatic areas, suggesting that these populations of Lu. intermedia present a degree of genetic diversity related to different habitats rather than season.16 In the present study, we have assessed by polymerase chain reaction–random amplified polymorphic DNA (RAPDDNA) analysis the magnitude and pattern of genetic differentiation between geographically separated populations of Lu. intermedia, which are involved in two specific transmission cycles of L. (V.) braziliensis genotypes in Espírito Santo.9 Analysis by PCR-RAPD17,18 has been widely used in genetic population studies of insects, including those incriminated as vectors of parasitic disease.19,20 Different studies have demonstrated that this is an efficient and sensitive method with the advantage that prior information about the genomic organization is not necessary. The RAPD technique is characterized by showing the variability of a large number of anonymous loci, theoretically distributed through the whole genome. The limitations associated with RAPD markers are well known and include variable reproducibility and a dominant mode of inheritance. However, a properly performed RAPD analysis is a useful and reliable tool for studying the ecology and genetic structure of populations. It has detected greater genetic diversity within species,20–24 and can help estimate the structure of Lu. intermedia populations circulating in ACL-endemic areas.

INTRODUCTION American cutaneous leishmaniasis (ACL) caused by Leishmania (Viannia) braziliensis is endemic and widely distributed in the state of Espírito Santo, Brazil, where there has been a consistent increase in the number of recorded cases in recent years.1 The ecology of ACL in Espírito Santo has been previously described.2–4 Transmission occurs in areas at medium and lower elevations (50–750 meters above sea level). The disease-endemic foci occur in small-scattered settlements, near heavily deforested mountains areas that show few remnants of the original vegetation. These rural villages are made up of well-dispersed houses with spaces between houses occupied by coffee and banana plantations. This disease is also found in old established communities near important cities on the Atlantic Coast. The armadillo (Euphractus sexcinctus) and the paca (Agouti paca) are the presumed reservoir hosts in the sylvatic cycle of L. (V.) braziliensis (Falqueto A and others, unpublished data), whereas dogs and possibly equines2,3 are implicated as domestic reservoirs of infection for humans in a number of foci.2–4 Leishmania (V.) braziliensis has been associated with a number of different sand fly species.5 Lutzomyia (Nyssomyia) intermedia (Lutz & Neiva) (Diptera, Phlebotominae) is probably the main vector of L. (V.) braziliensis in southeastern Brazil; Lu. migonei and Lu. whitmani are of secondary importance.6–8 This Leishmania species represents a polymorphic population9 and the different genotypes probably interact with several vector species, with implications for leishmaniasis control or intervention. These interactions might represent longstanding evolutionary relationships between sand flies and Leishmania, in which taxa of parasites and vectors are connected by unique behavioral10–13 or molecular

MATERIALS AND METHODS Collection sites and sand fly captures. Sand flies were collected with in a manual aspirator in two localities in Espírito Santo, Brazil (Afonso Claudio: 20°11.345⬘S, 41°03.781⬘W, altitude ⳱ 740 meters and Viana: 20°23.202⬘S, 40°27.412⬘W, altitude ⳱ 28 meters) (Figure 1) that are separated by ap-

* Address correspondence to Elisa Cupolillo, Laboratório de Pesquisas em Leishmanioses, Departamento de Imunologia, Instituto Oswaldo Cruz, IOC/FIOCRUZ, Pavilhão Leônidas Deane, Sala 509, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ, Brasil, 21045-900. E-mail: [email protected]




FIGURE 1. Study areas in the municipalities of Afonso Cláudio and Viana, in Espírito Santo State, Brazil. BA ⳱ Bahia; MG ⳱ Minas Gerais; ES ⳱ Espírito Santo; RJ ⳱ Rio de Janeiro.

proximately 50 km. The studied areas were selected based on the differences in the biogeography and in the ecoepidemiology of ACL1 of those areas. Two sand flies collections in each geographic area were conducted, one in June 2002 and another in August 2002. A total of 120 female sand flies were identified as Lu. intermedia by dissecting the last two abdominal tergites, which were then individually cleared and mounted in Berlese’s solution according to the method of Ryan and others.25 Identification used the criteria proposed by Young and Duncan26 and Marcondes.27 The remainder of the abdomen, head, and thorax from each female was transferred to separate 1.5-mL tubes and immediately stored in liquid nitrogen until molecular analysis was performed. The specimens were coded according to the macrohabitat (Afonso Claudio and Viana) and microhabitat (domestic and peridomestic) habitat: Afonso Cláudio intradomestic (ACi), Afonso Cláudio peridomestic (ACp), Viana intradomestic (Vi), and Viana peridomestic (Vp). PCR amplifications and electrophoresis. Total DNA was extracted from individual Lu. intermedia using the protocol described by Collins and others.28 A set of six decameric primers (Table 1) in the Ready-To-Go RAPD Analysis beads kit (Amersham Biosciences, Piscataway, NJ, USA) was used in the PCR-RAPD reactions. These primers were selected based on the clarity and consistency of the resulting banding

TABLE 1 Random amplified polymorphic DNA primers and their characteristics according to the number of fragments and genotypes* observed

Primer sequence (5⬘→3⬘)

No. of genotypes

No. of fragments


78 76 90 93 79 80 93†

27 37 46 49 44 32 235

No. of fragments by locality and microhabitats (ACi, ACp, Vi, Vp)

18, 20, 17, 25 09, 23, 24, 27 30, 26, 34, 26 39, 37, 32, 33 27, 27, 16, 36 24, 16, 19, 25

* ACi ⳱ Afonso Cla´ udio intradomestic; ACp ⳱ Afonso Cla´ udio peridomestic; Vi ⳱ Viana intradomestic; Vp ⳱ Viana peridomestic. † Considering the analysis of the patterns obtained by the six primers together.

patterns.16 The PCR-RAPD analysis was carried out according to the manufacture’s protocol in a final volume of 12.5 ␮L. All PCR amplifications were carried out in a PTC-200 Peltier Termal Cycler (MJ Research Inc., Waltham, MA) programmed for one cycle of 5 minutes at 95°C, 45 cycles for one minute at 95°C, one minute at 42°C, and two minutes at 72°C, and a final extension for 10 minutes at 72°C. An aliquot (5 ␮L) of each amplification product was separated by electrophoresis on 12.5% polyacrylamide gels (GeneGel Excel 12,5/ 24; Amersham Biosciences). The gels were stained using the DNA silver staining kit (Amersham Biosciences). A 100basepair DNA Step Ladder (Promega, Madison, WI) was used as a molecular size standard. All PCRs were performed after resuspending the PCR beads in water, followed by adding individual genomic DNA. Thus, we expected equal concentrations of PCR reagents in each tube. Duplicate negative controls without DNA were included in each RAPD experiment to ensure that there was no contamination. Two-tothree PCR amplifications per individual were carried out to evaluate the reproducibility of RAPD bands. Positive controls were also included in each RAPD experiment using the DNA of Escherichia coli from two different strains (BL21 and Cla) provided with the kit. The PCR-RAPD patterns for the positive controls were used also as a marker for reproducibility and contamination. Results of an RAPD experiment with no matching with the expected profile for the positive controls, as indicated by the manufacturer, were discarded. Data analysis. The RAPD markers were analyzed taking in consideration the following assumptions: 1) RAPD alleles segregate in a Mendelian fashion, 2) bands of equal electrophoretic mobility are homologous, 3) different loci segregate independently, and 4) populations are in Hardy-Weinberg equilibrium. Each Lu. intermedia was scored at each locus as 1 (present) or 0 (absent) across all polymorphic loci to create a binary matrix. A locus was considered polymorphic if the frequency of the most common allele did not exceed 0.95. Only distinct bands from gel to gel were used to generate the matrix data set. The genetic diversity within and among groups were quantified by the Dice coefficient and Euclidian distance using NTSYS software.29 A phenetic tree was constructed using the neighbor-joining algorithm. Bootstrap re-sampling (n ⳱ 1,000) was performed to test the robustness of the dendrogram topology, considering groupings with values ⱖ 90. Cophenetic analyzes was conducted to test the representativeness of the phenogram. The distances (Euclidian) among the individuals representing the four populations were used to perform a principal coordinate analysis (PCA) on the molecular variation shown by the RAPD data to visualize the geometric relationships among Lu. intermedia individuals or populations. To test the statistical significance of the results obtained by the numerical analysis conducted in this study, two model matrixes30 were created representing the distinct localities and the distinct microhabitat. Mantel tests were used to assess the statistical significance of the matrix correlation between the model matrixes and those obtained by genetic similarities/distances or FST values. Statistical significance was then tested using 1,000 random permutations and normalized by the Mantel statistic Z option using MXCOMP (NTSYS).29 The genetic structure of the populations was estimated using the fixation index. This index is useful to determine ge-


netic differentiation when RAPD markers are used because it allows an objective comparison of the overall effect of population substructure among different organisms without involving details of allele frequencies or levels of heterozygosity observed. The FST31 adapted by Lynch and Milligan32 to dominant markers was used to examine the overall level of genetic divergence among subpopulations using the qualitative guidelines suggested by Wright31 for the interpretation of the results: FST 0–0.05 ⳱ low genetic differentiation, FST 0.05–0.15 ⳱ moderate genetic differentiation, FST 0.15–0.25 ⳱ high genetic differentiation, and FST > 0.25 ⳱ great genetic differentiation. The FST was calculated using TFPGA software.33 The ␪ST value34 was calculated by analysis of molecular variance (AMOVA)35 using the Arlequin software package.36 The ␪ST is analogous to FST and represents the population structure, therefore indicating the components of internal variance.

RESULTS RAPD-PCR profiles. All six primers used produced clearly discernable and reproducible bands. For these selected primers, the same bands were always amplified in subsequent PCR analyses. The DNA of 120 female sand flies identified as Lu. intermedia were amplified using six decameric primers. After the amplification and electrophoresis, it was possible to analyze 93 profiles specific for each individual analyzed: 25 from ACi, 29 ACp, 19 from Vi, and 20 from Vp. Most bands observed were between 300 and 1,500 basepairs (Figure 2). Ambiguous and indistinguishable bands were excluded; 235 clearly discernable bands were analyzed, of which 90.2% were polymorphic. The number of RAPD markers and genotypes detected varied according to the geographic origin and habitat of sand fly collections (Table 1). The greatest number of fragments (172) was detected in the Vp population, whereas the Vi population contained less amplification products (142). The six primers exhibited somewhat different degrees of heterozygosity among tested sand flies. Although primer 4 produced the greatest number of genotypes, primer 2 generated the least (Table 1). However, only the RAPD-PCR profiles obtained after amplification by the primer 6 could be correlated with the four sand fly subpopulations studied (Figure 2), with each microhabitat group-

FIGURE 2. Random amplified polymorphic DNA–polymerase chain reaction profiles (12.5% polyacrylamide gel) after amplification of Lutzomyia intermedia DNAs by primer 6. Each lane contains amplification products for an individual sand fly. MM ⳱ molecular mass marker (100-basepair [bp] ladder). ACi ⳱ Afonso Cláudio intradomestic; ACp ⳱ Afonso Cláudio peridomestic; Vi ⳱ Viana intradomestic; Vp ⳱ Viana peridomestic.


ing together with the corresponding locality of collection. Numerical analyses performed using only the data obtained by primer 6 was in agreement with this observation (data not shown). None of the RAPD fragments of the Lu. intermedia populations studied was group specific. The frequencies of the bands varied in each population, but 21% of the Viana individuals exhibit a common band (450 basepairs) generated by primer 4 that was not observed in individuals from Afonso Claudio. In contrast, only 13% of individuals from Afonso Claudio had a common band (900 basepairs) produced by primer 5 that was not observed in the Viana population (data not shown). A 400-bp RAPD fragment generated by primer 5 was observed in almost all Lu. intermedia specimens examined, except in 5% of individuals from Vp. Genetic diversity within and among populations. Altogether, 235 discernable bands were used to estimate the level of genetic diversity within Lu. intermedia populations. The RAPD markers were transformed in a binary matrix for calculating the level of similarity between the 93 individuals analyzed. The level of similarity determined by the Dice’s coefficient ranged from 0.8 to 0.96 in Vi, from 0 to 0.86 in Vp, from 0.46 to 1 in ACi, and from 0.3 to 0.96 in ACp (Figure 3). Analysis of the scale in Figure 3 shows a lower level of genetic diversity within the population from Vi, compared with that within the population from Vp. This difference was not observed between the two populations from Afonso Cláudio, ACi and ACp, with both populations exhibiting almost the same range of genetic distance. To help resolve genetic relationships among populations, the binary matrices derived from the RAPD markers were used to calculate both similarity and distance indexes by Dice and Euclidian coefficients, respectively. The similarity/ distance matrixes were transformed into dendrograms using the neighbor-joining algorithm for grouping the individuals. The representativeness of the dendrograms was tested by cophenetic analysis. The dendrogram obtained applying the coefficient of Dice produced a good cophenetic value (r ⳱ 0.77), but the phenogram built with the Euclidian distance values and the neighbor-joining algorithm showed the highest value (r ⳱ 0.922). This dendrogram showed that the Lu. intermedia specimens were clustered in nine groups, with a strong bootstrap support (ⱖ 90). All of these groups had a tendency to cluster individuals of the same microhabitat (data not shown). Principal coordinate analysis was performed to obtain further insights into the genetic relationships among Lu. inter-

FIGURE 3. Range of genetic diversity observed in the Lutzomyia intermedia populations by analysis of the level of similarity by applying Dice’s coefficient to the random amplified polymorphic DNA data. ACp ⳱ Afonso Cláudio peridomestic; ACi ⳱ Afonso Cláudio intradomestic; Vp ⳱ Viana peridomestic; Vi ⳱ Viana intradomestic.



FIGURE 4. Principal coordinate analyzes of individuals within subpopulations of Lutzomyia intermedia plotted in three principal axes (variation observed ⳱ 14.5%). ACi, Afonso Cláudio intradomestic; ACp, Afonso Cláudio peridomestic; Vi, Viana intradomestic; Vp, Viana peridomestic.

media populations. The scores for the four populations were plotted along with the first three axes, which accounted for only 14.5% of the total variance. It is interesting to observe that even representing a low level of variance, there was a spatial distribution for each population, except for individuals from Vp that dispersed along the three axes (Figure 4). This is in accordance with the result presented in the Figure 3, which shows that the Vp population is more heterogeneous. The statistic significance of genetic differences observed among the populations was evaluated by the Mantel test. In this analysis, the difference observed between the populations corresponding to the localities (Viana and Afonso Claudio) and among the four populations corresponding to the localities and microhabitats was considered. The values obtained showed that the differences among the populations, considering either the localities or the microhabitat, are statistically significant (P > 0.001). Genetic structure of the populations. For determining if the genetic differences estimated for the sand flies are reflecting gene flow among insect populations, fixation index (FST) estimates were calculated according to Lynch and Milligan32 and AMOVA approaches35(Table 2). The values of FST obtained by both methods were equivalent. The AMOVA results demonstrate that a large proportion of total variation was observed among individuals (90.89%), but only a modest proportion of variation would be attributed to either differences observed among populations (0.89%) or

among groups within population (8.22%) (Table 2), which reflects that populations of Lu. intermedia were not distinct units but groups of individuals in regular association. The values of FST obtained by the Lynch and Milligan32 approach were variable, which showed low genetic differentiation among populations (AC × V ⳱ 0.081 ± 0.007). However, when analysis was performed between populations Afonso Cláudio microhabitats, the analysis showed moderate genetic differentiation (ACi × ACp ⳱ 0.149 ± 0.02). Viana microhabitats showed high genetic differentiation (Vp × Vi ⳱ 0.177 ± 0.015). These values corroborated with those obtained by AMOVA,35 which showed higher values between microTABLE 2 Analysis of molecular variance of populations and individuals of Lutzomyia intermedia*

Source of variation

Among populations (AC × V) Among groups within populations (ACi, ACp, Vi, Vp) Among individuals Total





2 89 92

113.572 1,632.710 1,810.473

Variation components (% of variation)


0.180 (0.89)


1.659 (8.22) 18.345 (90.89) 20.184

0.164 0.008 –

* df ⳱ degrees of freedom; SS ⳱ sum of squares. For definitions of other abbreviations, see Table 1.


habitats compared with geographic region (Table 2). These results are in agreement with those obtained by PCA (Figure 4), which shows groups formed by individuals from the same microhabitat but does not allow the distinction of groups according to the geographic locality.

DISCUSSION Hypotheses of distinct transmission cycles of leishmaniasis occurring in the same endemic area have been postulated.37,38 Despite evidence of genetic differentiation between sand flies caught in sylvatic and domestic or peridomestic habitats, genetic similarities among samples collected in a restricted focus of AVL in Venezuela indicated the existence of a genetically homogenous Lu. longipalpis population that extended from houses to closely located natural habitat.39 This observation indicated not only the potential of sand fly species to transport Leishmania parasites between enzootic and domestic cycles, but also that new foci may be initiated by infected sand flies moving between both habitats.40 Previous studies demonstrated that analysis of Lu. intermedia populations using RAPD markers, which produces higher genetic variability than multi-locus enzyme electrophoresis, showed that the genetic distance between populations of intradomestic and peridomestic habitats was insignificant, but the specimens collected in the sylvatic environment exhibited a significant genetic divergence when compared with those from the peridomestic and/or intradomestic environment.16 This study was conducted in a focus of ACL in the state of Rio de Janeiro where there is almost no genetic differentiation among L. braziliensis parasites. In the present study, we analyzed by RAPD-PCR Lu. intermedia populations collected in distinct microevironments and macroenvironments that represented ACL foci where two separate groups of L. braziliensis genotypes were considered as being involved with specific transmission cycles of this parasite.9 The RAPD data showed that the number of polymorphic bands did not vary significantly according to the geographic origin of populations or microhabitats (Table 1), and there was no RAPD marker specific to any one of the studied populations. The most frequent RAPD fragment had a frequency of 38%, which indicated mixture of subpopulations, and consequently, the absence of fixed alleles in the populations. Clark and Lanigan41 have reported that local populations will not necessarily show fixed differences. The degree of genetic structure between Afonso Cláudio and Viana populations was low (0.0808), which indicated that geographic separation of these populations was a recent event. It is possible that the differentiation observed between these populations of Lu. intermedia is being influenced by several natural barriers to flight. These localities, which are located at approximately 700 meters and 30 meters above sea level, respectively, are separated by approximately 150 km. Analysis of the distribution of sand flies at different altitudes provided evidence that Lu. intermedia is the main vector of L. (V.) braziliensis in Espírito Santo, but Lu. whitmani and Lu. migonei might act as secondary vectors of the parasite in rural localities.4 The influence of altitude was also noted in some structure sizes of Lu. intermedia.42 The FST values estimated between microhabitats were higher than between sand fly populations from localities sepa-


rated by 150 km. This result corroborated other studies of population genetics of insect vectors, which demonstrated that the populations are homogeneous in a radius of 20 km and that a certain degree of structuring is observed in populations encountered in a zone of approximately 1 km.39,43–45 Clustering analysis by either the hierarchical or ordination method indicate a tendency of Lu. intermedia individuals to form clusters according to the microhabitat of collection (intradomestic and peridomestic). It is clear that the Lu. intermedia population circulating in the Viana municipality is more heterogeneous than that circulating in Afonso Claudio. A factor that may be contributing for the genetic diversity observed in both population and the differences between the genetic diversity observed in each population may be the difference in the time of human colonization of the two localities (200 and 80 years ago for Viana and Afonso Cláudio respectively), which may reflect differences in the behavior of populations of Lu. intermedia. Although there was a tendency of individuals collected in the same habitat to cluster in the same phenetic group, this fact does not necessarily indicate genetic differentiation between the populations. Individuals collected in the domestic environment might exhibit phenotypes associated with, for example, phototropism and alimentary behaviors, thus influencing the distribution of the insects in this environment. Even though sand flies take a blood meal preferentially in domestic animals, they will be attracted to the intradomestic habitat, as indicated by the low population substructuring of Lu. intermedia analyzed. This attraction is probably maintaining and/or increasing the prevalence of the human infection in the studied areas. The genetic analysis of isolates of L. (V.) braziliensis collected in the two disease-endemic areas (Afonso Claudio and Viana) studied herein16 demonstrated that the degree of genetic diversity of L. (V.) braziliensis from Afonso Cláudio is higher than that observed among parasites isolated from both patients and dogs in Viana. If one considers that F estimates (FST) for Lu. intermedia specimens collected at Afonso Cláudio indicated the existence of a genetically more homogeneous population than those from Viana, other sand fly species (such as Lu. whitmani) may act as vectors involved in the transmission of L. (V.) braziliensis in that endemic area, therefore influencing the genetic diversity of the parasites found in that region.9 A search for suspected vectors was conducted in the municipality of Afonso Cláudio in the midwest region of Espírito Santo (a sample of 13,363 specimens belonging to 28 species was collected). The five most abundant human-biting species collected were Lu. intermedia (24.3%), Lu. migonei (22.3%), Lu. whitmani (15.4%), Lu. fischeri (14.9%), and Lu. monticola (5.8%).4 Gene flow between Lu. intermedia and Lu. whitmani could have involved exchange of behavioral and other traits of epidemiologic importance in that region.46 Our results indicated that different ACL regions might have distinct transmission patterns. The geographic structuring observed for some L. braziliensis genotypes could reflects specific parasite-sand fly vector interactions (such as coevolution between molecules of the sand fly midgut and the parasite external surface)15 and not geographic isolation of the parasite, as observed in Trypanosoma rangeli.47 In localities where there are several sand fly species participating in the transmission cycle of ACL, e.g., Afonso Cláudio, there is



a tendency of the population of parasites to be more heterogeneous. Conversely, in areas where Leishmania transmission is associated with only one sand fly species, the parasite population is more homogeneous, as has been observed in Viana.9 Furthermore, in areas where the population of the vector, circulating in the different habitats, is more homogeneous, it has not been observed genetic polymorphism in the parasite population.9,16 The moderate level of genetic structuring observed in Lu. intermedia populations in different habitats (intradomestic and peridomestic) in Afonso Cláudio, a rural area, probably reflect extension of the domestic transmission cycle in this areas. Conversely, the high level of genetic structuring of Lu. intermedia populations in the different habitats of a periurban area (Viana) may reflect independence between the domestic and the peridomestic cycle. These observations support increasing genetic divergence for Lu. intermedia to adapt to changes in their original forested habitats through decades in the same area with important public health implications.






Received April 11, 2006. Accepted for publication August 24, 2006. Financial support: This study was supported by PRONEX/CNPq (National Council for Scientific and Technological Development of the Ministry of Science and Technology, Brazil) and the Carlos Chagas Filho Research Foundation of the State of Rio de Janeiro (FAPERJ). Elisa Cupolillo and Gabriel Grimaldi Jr. are CNPq fellow researchers, Leonardo de Souza Rocha is a PhD student in the Program in Molecular and Cell Biology/FIOCRUZ and is sponsored by CNPq. Authors’ addresses: Leonardo de Souza Rocha, Gabriel Grimaldi Jr, and Elisa Cupolillo, Laboratório de Pesquisas em Leishmanioses, Departamento de Imunologia, Instituto Oswaldo Cruz, IOC/ FIOCRUZ, Pavilhão Leônidas Deane—sala 509, Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ, Brasil, 21045-900, Telephone: 5521-3865-8177, Fax: 55-21-2209-4110, E-mails: [email protected], [email protected], and [email protected] Aloísio Falqueto and Claudiney Biral dos Santos, Unidade de Medicina Tropical, Universidade d’Espírito Santo, Av. Marechal Campos 1468, Vitória, ES, Brasil, 29040-090, E-mails: [email protected] and [email protected]

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