Molecular evidence of sustained urban malaria transmission in Amazonian Brazil, 2014-2015.

The relative contribution of imported vs. locally acquired infections to urban malaria burden remains largely unexplored in Latin America, the most urbanised region in the developing world. Here we use a simple molecular epidemiology framework to examine the transmission dynamics of Plasmodium vivax in Mâncio Lima, the Amazonian municipality with the highest malaria incidence rate in Brazil. We prospectively genotyped 177 P. vivax infections diagnosed in urban residents between June 2014 and July 2015 and showed that local parasites are structured into several lineages of closely related microsatellite haplotypes, with the largest genetic cluster comprising 32% of all infections. These findings are very unlikely under the hypothesis of multiple independent imports of parasite strains from the rural surroundings. Instead, the presence of an endemic near-clonal parasite lineage circulating over 13 consecutive months is consistent with a local P. vivax transmission chain in the town, with major implications for malaria elimination efforts in this and similar urban environments across the Amazon.

Human toxocariasis: diagnosis, worldwide seroprevalences and clinical expression of the systemic and ocular forms.

Although human toxocariasis ranks among the most common zoonotic infections worldwide, it remains relatively unknown to the public. The causal agents are the nematode parasites Toxocara canis and T. cati, whose definitive hosts are dogs and cats, respectively. When embryonated eggs are accidentally ingested by humans, larvae hatch in the small intestine, penetrate the intestinal wall and migrate, via the bloodstream, to the liver, lungs, muscles, eye and central nervous system. Although most human infections are asymptomatic, two well-defined clinical syndromes are classically recognised: visceral larva migrans (a systemic disease caused by larval migration through major organs) and ocular larva migrans (a disease limited to the eyes and optic nerves). Two less-severe syndromes have recently been described, one mainly in children (covert toxocariasis) and the other mainly in adults (common toxocariasis). Here, the current laboratory diagnosis, epidemiology and main clinical features of both the systemic and ocular forms of human toxocariasis are reviewed. New developments in serological diagnosis are described, the available seroprevalence data are analysed, and the results of relevant clinical studies that have been published over the last decade are explored, to provide an updated overview of this neglected but highly prevalent human infection.

Population dynamics of genetically diverse Plasmodium falciparum lineages: community-based prospective study in rural Amazonia.

Temporal changes in the prevalence of antigenic variants in Plasmodium falciparum populations have been interpreted as evidence of immune-mediated frequency-dependent selection, but evolutively neutral processes may generate similar patterns of serotype replacement. Over 4 years, we investigated the population dynamics of P. falciparum polymorphisms at the community level by using 11 putatively neutral microsatellite markers. Plasmodium falciparum populations were less diverse than sympatric P. vivax isolates, with less multiple-clone infections, lower number of alleles per locus and lower virtual heterozygosity, but both species showed significant multilocus linkage disequilibrium. Evolutively neutral P. falciparum polymorphisms showed a high turnover rate, with few lineages persisting for several months in the population. Similar results had previously been obtained, in the same community, for sympatric P. vivax isolates. In contrast, the prevalence of the 2 dimorphic types of a major antigen, MSP-2, remained remarkably stable throughout the study period. We suggest that the relatively fast turnover of parasite lineages represents the typical population dynamics of neutral polymorphisms in small populations, with clear implications for the detection of frequency-dependent selection of polymorphisms.

In Silico Identification of Novel Biomarkers and Development of New Rapid Diagnostic Tests for the Filarial Parasites Mansonella perstans and Mansonella ozzardi.

Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans, M. ozzardi, M. rodhaini and M. streptocerca. Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequences have been used as highly sensitive biomarkers for filarial nematodes. We have developed a bioinformatic pipeline to mine Illumina reads obtained from sequencing M. perstans and M. ozzardi genomic DNA for new repeat biomarker candidates which were used to develop loop-mediated isothermal amplification (LAMP) diagnostic tests. The M. perstans assay based on the Mp419 repeat has a limit of detection of 0.1 pg, equivalent of 1/1000th of a microfilaria, while the M. ozzardi assay based on the Mo2 repeat can detect as little as 0.01 pg. Both LAMP tests possess remarkable species-specificity as they did not amplify non-target DNAs from closely related filarial species, human or vectors. We show that both assays perform successfully on infected human samples. Additionally, we demonstrate the suitability of Mp419 to detect M. perstans infection in Culicoides midges. These new tools are field deployable and suitable for the surveillance of these understudied filarial infections.

Evolution of allelic dimorphism in malarial surface antigens.

The extensive sequence variation in most surface antigens of Plasmodium falciparum is one of the major factors why clinical immunity to malaria develops only after repeated infections with the same species over several years. For some P. falciparum surface antigens, all observed alleles clearly fall into two allelic classes, with divergence between classes dwarfing divergence within classes. We discuss the ways in which such allelic dimorphism deviates from the expected shape of the genealogy of genes under either neutral evolution or standard balancing selection, and present a simple test, based on coalescent theory, to detect this deviation in samples of DNA sequences. We review previous hypotheses for the origin and evolution of allelic dimorphism in malarial antigens and discuss the difficulties of explaining the available data under these proposals. We conclude by offering several possible classes of explanations for allelic dimorphism, which are worthy of further theoretical and empirical exploration.

Diversity in the thrombospondin-related adhesive protein gene (TRAP) of Plasmodium vivax.

We analyzed 22 clinical isolates of Plasmodium vivax from Thailand and 17 from Brazil to investigate the extent of sequence variation in the thrombospondin-related adhesive protein of Plasmodium vivax (PvTRAP), a homologue of P. falciparum TRAP (PfTRAP) which has been considered to be a promising vaccine candidate. In total 54 haplotypes were identified from 73 distinct gene clones. Coexistence of different PvTRAP in circulation occurred in 10 and 13 isolates from Thailand and Brazil, respectively. Forty out of 48 substituted nucleotides are non-synonymous changes. Most of the substituted residues reside in the von Willebrand factor type A-domain (region II), a sulfated glycosaminoglycan-binding domain (region III) and a proline-rich region (region IV). All nucleotide substitutions are dimorphic. Two haplotypes from Thailand contain an inserted sequence encoding aspartic acid-serine-proline in the proline-rich region. Sequence analysis has revealed that nucleotide diversity in PvTRAP is low although Brazilian isolates display a higher degree of variation than those from Thailand. Phylogenetic construction using the neighbor joining method has shown that most of the Thai and the Brazilian isolates appear to be mainly clustered into distinct groups. Significantly greater than expected values of the mean number of non-synonymous (d(n)) than synonymous (d(s)) nucleotide substitutions per site were observed in regions II and III of PvTRAP. Analysis of the published PfTRAP sequences has shown a similar finding in regions II and IV suggesting that positive selection operates on the regions. Hence, different regions in PvTRAP and PfTRAP could be under different pressures in terms of immune selection, structural and/or functional constraints.

Selection and genetic drift of polymorphisms within the merozoite surface protein-1 gene of Plasmodium falciparum.

Intragenic recombination in the merozoite surface protein-1 gene (Msp-1) of Plasmodium falciparum is a major mechanism for allelic variation among natural parasite populations. The frequency of recombination depends on the intensity of transmission in the vector mosquito. In the present study, linkage disequilibrium between polymorphic 'loci' in the 5'- and 3'-regions of Msp-1 was examined in parasite populations from Brazilian Amazon and southern Vietnam and compared with that in a Thai population previously reported. The R2 test identified clusters of linkage disequilibria between the 5'- and 3'-regions, which are different among the three populations. However, the overall strength of linkage disequilibria was stronger in Brazil, a hypoendemic area, than in Vietnam and Thailand, mesoendemic areas, suggesting that linkage disequilibrium in Msp-1 inversely correlates with the intensity of transmission. To investigate possible mechanisms for linkage disequilibrium in Msp-1, we applied the Fst index, which measures the inter-population variance in allele frequency, to 'loci' in Msp-1 among the three populations. The Fst test identified two distinct regions with respect to inter-population allele frequency in Msp-1: one for highly divergent 'loci' in the 5'-region and the other for non-divergent 'loci' in the 3'-region. These results suggest that genetic drift is not the sole mechanism for linkage disequilibrium, but selection operates on 'loci' in the 3'-region in hypo- and mesoendemic areas of malaria.

Meiotic recombination, cross-reactivity, and persistence in Plasmodium falciparum.

We incorporate a representation of Plasmodium falciparum recombination within a discrete-event model of malaria transmission. We simulate the introduction of a new parasite genotype into a human population in which another genotype has reached equilibrium prevalence and compare the emergence and persistence of the novel recombinant forms under differing cross-reactivity relationships between the genotypes. Cross-reactivity between the parental (initial and introduced) genotypes reduces the frequency of appearance of recombinants within three years of introduction from 100% to 14%, and delays their appearance by more than a year, on average. Cross-reactivity between parental and recombinant genotypes reduces the frequency of appearance to 36% and increases the probability of recombinant extinction following appearance from 0% to 83%. When a recombinant is cross-reactive with its parental types, its probability of extinction is influenced by cross-reactivity between the parental types in the opposite manner; that is, its probability of extinction after appearance decreases. Frequencies of P. falciparum outcrossing are mediated by frequencies of mixed-genotype infections in the host population, which are in turn mediated by the structure of cross-reactivity between parasite genotypes. The three leading hypotheses about how meiosis relates to oocyst production lead to quantitative, but no qualitative, differences in these results.

The assessment of antibody affinity distribution by thiocyanate elution: a simple dose-response approach.

We describe a simple dose-response approach to assess the affinity distribution of polyclonal antibodies. The proportion of antigen-specific antibodies dissociated by increasing concentrations of the mild chaotropic agent ammonium thiocyanate (NH4SCN) was measured by enzyme immunoassay, and the distribution of tolerances to this agent was presented in a histogram form. Such 'tolerance distribution', which is analogous to that described in classical dose-response bioassays, is proposed as a representation of the actual antibody affinity distribution. To test this approach, we assessed affinity maturation patterns of anti-Plasmodium falciparum IgG antibodies in paired sera obtained from 22 malaria patients during the acute infection and convalescence. We obtained patterns of antibody affinity distributions consistent with those previously described in immunization experiments with the aid of more complex laboratory and computational approaches. Therefore, we suggest the thiocyanate elution technique as an alternative method for rapid assessment of affinity distributions of polyclonal antibodies elicited against complex antigens, readily applicable to large number of serum samples.

Unstable hypoendemic malaria in Rondonia (western Amazon region, Brazil): epidemic outbreaks and work-associated incidence in an agro-industrial rural settlement.

A longitudinal study was conducted from January 1991 to January 1992 on the Urupa farm, a rural agro-industrial forestry settlement in Rondonia state (Western Amazon Region, Brazil) to define the parasitologic and clinical profile of malaria. Three cross-sectional, parasitologic, and clinical surveys were performed. In the intervals between surveys, malaria cases were monitored by twice a week medical visits to the farm and permanent local surveillance. The population of residents was approximately 170 and was characterized by high mobility. The slide positive rates found in the cross-sectional surveys were 0.5, 4.2 and 2.1, respectively, for the total population (Plasmodium vivax plus P. falciparum). Spleen rate values in children 2-9 years old were always less than 1%. However, this basically hypoendemic malaria situation was unstable, with occurrence of a typical epidemic outbreak at the end of the dry season. The total number of malaria cases recorded from January to December 1991 was 163, giving an annual parasite index of 970 per 1,000 inhabitants. However, sex and age distribution of cases showed rare incidence of malaria in infants and low incidence in children less than the age of 10. Male adults 16-40 years of age represented the main risk group. The observed clustering of cases allowed us to identify the place of work as a factor responsible for high incidence of malaria among adults. The general epidemiologic profile indicated that indoors transmission of malaria by the local Anopheles vector was low or absent.

The isotype composition and avidity of naturally acquired anti-Plasmodium falciparum antibodies: differential patterns in clinically immune Africans and Amazonian patients.

A critical role has been proposed for cytophilic IgG1 and IgG3 subclass antibodies and monocytes and macrophages in antimalarial immunity. Here we compared the isotype composition and avidity of naturally acquired antibodies, as measured by enzyme immunoassay against a detergent-soluble extract of Plasmodium falciparum schizonts, in clinically immune Senegalese adults (n = 33) and semi-immune, adult Amazonian patients (n = 25). Plasma were collected during an acute symptomatic P. falciparum attack and two months later, and in the absence of recrudescence or reinfection. Specific IgG, IgM, IgA, and IgG subclass antibodies were assessed. The results are summarized as follows: 1) high-avidity cytophilic antibodies predominated in clinically immune Senegalese subjects; 2) acutely ill Amazonian patients produced high levels of low-avidity cytophilic antibody; 3) such a response was shortlived, since two months later, the concentrations of cytophilic antibodies were significantly lower; 4) however, affinity maturation of IgG antibodies was observed in Amazonian patients two months after the acute malaria attack. A considerable proportion (35-46%) of anti-P. falciparum IgG1 antibodies produced by African and Amazonian patients was shown to recognize periodate-sensitive carbohydrate epitopes. The potential impact of these findings on the design and evaluation of antimalarial vaccines is discussed.

Longitudinal study of naturally acquired humoral immune responses against the merozoite surface protein 1 of Plasmodium vivax in patients from Rondonia, Brazil.

A longitudinal study on the naturally acquired humoral immune responses against the merozoite surface protein 1 of Plasmodium vivax (PvMSP-1) was performed in malaria patients from the Brazilian Amazon region of Rondonia. We have previously cloned and expressed a recombinant protein, ICB2-5, that encodes 508 amino acids from the N-terminal portion of the PvMSP-1 protein. This affinity-purified polypeptide was tested by an enzyme-linked immunosorbent assay in a one-year longitudinal study using sera from 34 patients who had at least one malaria infection during the study period. The results demonstrated that more than 90% of the sera from patients having experienced more than three previous malaria infections contained antibodies to ICB2-5 at the time of a new clinical episode. Unexpectedly, more than half of these multiple-infected patients had an antibody response to ICB2-5 in which the predominant isotype was IgM. In contrast, more than 83% of the sera from these same patients contained predominantly IgG antibodies against total blood-stage antigen preparations. To determine if these results were due to the lack of boosting against this portion of the PvMSP-1 molecule, the presence of IgG antibodies to ICB2-5 in the sera from 11 patients who had consecutive malarial episodes during the study year was investigated. Five of these eleven patients failed to produce IgG antibodies to ICB2-5 even after 1-3 infections. Thus, these results suggest that no boosting against this region of the PvMSP-1 molecule was achieved by natural infections among these patients.

Characterization of naturally acquired human IgG responses against the N-terminal region of the merozoite surface protein 1 of Plasmodium vivax.

The primary structure of the merozoite surface protein 1 of Plasmodium vivax (PvMSP-1) revealed the existence of conserved and polymorphic blocks of the protein among different Plasmodium species. To characterize the naturally acquired IgG antibody responses to the PvMSP-1 molecule, the entire N-terminal portion of this protein was expressed as 10 overlapping glutathione S-transferase fusion proteins. The affinity-purified recombinant products were tested by enzyme-linked immunosorbent assay and Western blot against the sera of malaria patients from the state of Rondonia, Brazil. We found that the majority of these sera did not contain IgG antibodies recognizing recombinant proteins expressing exclusively interspecies conserved blocks of the molecule. In contrast, a high proportion of these same sera reacted against recombinant products expressing interspecies polymorphic regions of this protein. The poor B cell immunogenicity of the interspecies conserved blocks of the PvMSP-1 molecule most likely reflects important and unknown structural or functional features of these regions.

Allelic diversity at the merozoite surface protein-1 locus of Plasmodium falciparum in clinical isolates from the southwestern Brazilian Amazon.

Nucleotide sequences of each variable block in the Plasmodium falciparum merozoite surface protein-1 gene (PfMSP-1) may be grouped into one of two or three possible allelic types, named after the reference isolates MAD20, K1, and RO33. Allelic diversity at this locus basically results from different combinations of allelic types in variable blocks. We used a polymerase chain reaction (PCR)-based strategy to type the variable blocks 2, 4a, 4b, and 10 of the PfMSP-1 gene of P. falciparum isolates from 54 symptomatic malaria patients living in Rondonia, a hypoendemic area in the southwestern Brazilian Amazon. Ten different PfMSP-1 gene types, defined as unique combinations of allelic types in variable blocks, were identified among the 54 isolates. Twenty-one isolates (39%) harbored more than one gene type and two had at least three genetically distinct clones. Hybrid sequences, with a MAD20-type sequence in the 5' segment (4a) and a K1-type sequence in the 3' segment (4b), were quite common in block 4. Direct sequencing of block 4 PCR products revealed a new putative recombination site in four isolates. In contrast with previous studies, the observed distribution of gene types does not deviate significantly from that expected under the null hypothesis of random association between allelic types detected in each variable block. These contradictory data are discussed with reference to the immunoepidemiologic features prevailing in distinct malaria-endemic areas.

Hypoendemic malaria in Rondonia (Brazil, western Amazon region): seasonal variation and risk groups in an urban locality.

A longitudinal epidemiologic survey (1989-1991) plus a cross-sectional parasitologic, clinical, and sociodemographic survey (July-October 1990) were conducted in Candeias do Jamary, a village with approximately 7,000 inhabitants in Rondonia, Brazil. Analysis of the results revealed hypoendemic malaria with a complex epidemiology. Plasmodium vivax predominated over P. falciparum infections while infections with P. malariae were absent. Malaria is present throughout the year but was clearly seasonal with epidemic outbreaks in the dry season from June to August. Malaria prevalence was lower in children less than 10 years of age and significantly higher in young adult males, which represent the high-risk group. The incidence of locally acquired infections (autochthonous cases) was significantly lower in the rainy season as compared with the dry season. This is not true with respect to heterotochthonous (imported) malaria cases, that is, malaria acquired elsewhere by Candeias residents, most of whom are male adults working outside the town. In both cases, however, the age and sex distribution of prevalence and its relationship with occupational activities indicate a predominance of outdoor transmission. The results of the cross-sectional survey are in agreement with those of the longitudinal passive survey and, in addition, disclose the absence of asymptomatic infection.

Antibody response against Plasmodium falciparum exoantigens and somatic antigens: a longitudinal survey in a rural community in Rondônia, western Brazilian Amazon.

Three clinical and sero-epidemiological cross-sectional surveys involving 50 subjects were performed at six-month intervals in Urupá, a rural community characterized by unstable malaria transmission, situated in Rondônia State, Western Brazilian Amazon. Between the surveys, a clinically and parasitologically passive surveillance was established in this community and 48 malaria attacks (28 due to Plasmodium falciparum and 20 due to Plasmodium vivax) were recorded in this cohort of 50 subjects. Serum samples were collected at each survey and tested by enzyme immunoassay (ELISA) for IgG, IgG subclass and IgM antibodies against P. falciparum exoantigens isolated from culture supernatants and detergent-soluble somatic antigens. As expected, both anti-malarial IgG and IgM antibody titres were shown to rise after a malaria outbreak observed during the follow-up period. Nevertheless, in marked contrast with the profile of anti-malarial IgG subclasses described for semi-immune Africans, in this Amazonian community IgG2 antibodies (that are non-cytophilic) against both antigens were shown to predominate over other IgG subclasses. Such overall predominance of IgG2 subclass titres was statistically significant concerning exoantigens, but was of borderline significance in relation to IgG1 antibodies against somatic antigens (p = 0.052). Moreover, highly variable patterns of boosting were observed in antibody responses against both antigens among the patients who suffered P. falciparum malaria attack during the study.

Humoral immune response to the 72 kDa heat shock protein from Plasmodium falciparum in populations at hypoendemic areas of malaria in western Brazilian Amazon.

The heat-shock protein Pf72/Hsp70-1 from the human malaria parasite Plasmodium falciparum has been suggested as a potential candidate antigen for a multivalent vaccine. We have investigated the prevalence and levels of IgG antibodies to the recombinant protein PfR44, derived from Pf72/Hsp70-1, in individuals from different age groups living in Candeias do Jamari, an Amazonian town characterized by unstable and hypoendemic malaria transmission. Blood were collected from a household-based random sample comprising 241 people and the sera were comparatively tested against recombinant antigen PfR44 and a detergent-soluble extract of P. falciparum (PfAg-T). The prevalence and levels of IgG antibodies to both recombinant and total P. falciparum antigens were positively correlated with cumulative exposure to malaria, as estimated by the age of the individuals and the duration of their stay in the study area. Nevertheless, correlations between antibody responses to Pf72/Hsp70-1 and the acquisition of protective anti-malarial immunity could not be derived from our data.

Anaemia, iron deficiency and malaria in a rural community in Brazilian Amazon.

OBJECTIVE: To assess the incidence of anaemia, iron deficiency and malaria in a malaria-endemic community. DESIGN: Three consecutive cross-sectional surveys (A, B and C) of the whole population made at 6-month intervals and malaria surveillance between the surveys. SETTING: Urupá, a rural community in Western Brazilian Amazon. SUBJECTS: 133 people of all age groups present in at least two cross-sectional surveys. INTERVENTIONS: Anaemic patients received ferrous sulphate during 3 months. Patients parasitized by intestinal nematodes were given mebendazole and parasitologically proven Plasmodium falciparum and P. vivax malaria attacks were treated with quinine or chloroquine plus primaquine. RESULTS: Anaemia (haemoglobin concentrations [Hb] below the cut-off values proposed by the World Health Organization) was diagnosed in respectively 10.0% (13 of 130) subjects in survey A, 9.2% (10 of 109) in B and 29.7% (27 of 91) in C. Depleted iron stores [serum ferritin (SF) < 12 micrograms/l] were detected in 10.0% subjects in survey A, 10.1% in B but in only 8.8% subjects in survey C. Concomitant anaemia and low SF was detected in 5.4% subjects in survey A, 3.7% in B and 6.6% in C. Mean Hb from anaemic patients diagnosed and treated during the study (n = 17) raised 1.2 g/dl after iron therapy and most of them (13 of 17, 76.5%) became non-anaemic. The highest malaria transmission was observed between surveys B and C. People who suffered at least one malaria attack during this period (27 of 63) were at a slightly greater risk of subsequent anaemia (odds ratio = 2.85, 95% confidence interval 0.81-10.28). CONCLUSIONS: Both malaria and iron deficiency could be considered as important causes of anaemia in this population. SPONSORSHIP: Supported by grants from the UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases (no. 890245), the Ministére des Affaires Etrangeres, France, and from the Fundação de Amparo à Pesquisa do Estado de São Paulo (no. 92/1336-4). M.A.C. was supported by a doctoral fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico.

A simple reflectance microphotometer for dot immunobinding assay (dot-ELISA) quantification.

The use of a simple reflectance microphotometer adapted to quantify enzymatic dot immunobinding assays is described. The instrument is versatile and precise by clinical laboratory standards (i.e., 1.1% coefficient of variation for ten consecutive readings of a grey cardboard). The instrument provides a low-cost alternative to commercially available equipment for quantitation of dot-ELISA assays.

Effect of dietary iron on the course of Plasmodium berghei malaria in young rats.

Clinical and experimental evidence suggests that iron-deficient hosts are less susceptible to severe malaria and that iron supplementation aggravates infection. In the present study, 60 weanling Wistar rats were fed standard diets with different iron concentrations: 21 mg/kg (group 1), 45 mg/kg (group 2) and 113 mg/kg (group 3). Ferrous sulfate (FeSO4 x 7H2O) was added to the normal-iron and iron-supplemented diets (groups 2 and 3, respectively). Data are reported as mean +/- SEM. After 16 days of regimen, eight rats from each group were killed to measure serum iron concentration (SI) and transferrin saturation capacity (TSC). At this moment, rats from group 1 were underweight and their dietary intake was significantly lower than that of animals from the other groups. Severe iron deficiency (SI = 49.2 +/- 4.5 micrograms/100 ml and TSC = 8.3 +/- 0.7%) was observed in rats from group 1, while the animals from the other groups were iron-sufficient (group 2: SI = 186.5 +/- 28.5 micrograms/100 ml and TSC = 27.3 +/- 3.4%; group 3: SI = 137.3 +/- 18.2 micrograms/100 ml and TSC = 21.3 +/- 2.3%). Nine animals from each group were then infected with the malaria parasite Plasmodium berghei, whereas three animals from each group were used as noninfected controls. Parasitemias (% of infected red blood cells) peaked 7 days post-infection in animals from groups 2 and 3 (mean values of 2.4% and 1.7%, respectively), but in animals from group 1 parasitemias increased until the 9th day post-infection (mean at peak, 2.3%) and parasite clearance was significantly slower than in the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)