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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. [ABSTRACT FROM AUTHOR]

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.Heredity (2008) 100, 103–110; doi:10.1038/sj.hdy.6800887; published online 4 October 2006 [ABSTRACT FROM AUTHOR]

We have optimized a set of 14 polymorphic microsatellite markers for the human malaria parasite Plasmodium vivax, all of them consisting of either tri- or tetranucleotide repeats. These markers, whose polymerase chain reaction amplification conditions are identical, were used to screen 25 parasite isolates from malaria-endemic areas in Sri Lanka. The total number of alleles per locus ranged between 6 and 13 (average, 7.8), and expected heterozygosity ranged from 0.627 to 0.913 (average, 0.790). These markers are now being used to characterize the population structure of P. vivax in other endemic areas. [ABSTRACT FROM AUTHOR]

A critical role has been proposed for the switch from non-cytophilic IgG2 to cytophilic antibodies of IgG1 and IgG3 subclasses observed in the humoral immune responses to Plasmodium falciparum of some Africans. These Africans have acquired clinically immunity naturally, after several years of exposure to holo-endemic malaria. In the present study, the possibility that life-long exposure to low levels of malarial endemicity may be associated with changes in the IgG-subclass composition of antibodies to P. falciparum was investigated in a native Amazonian community. The subjects were 138 malaria-exposed but non-infected Karitiana Indians. In a separate investigation, the concentrations of IgG-subclass antibodies in acutely ill patients with severe malaria ( N = 22) were compared with those in age- and sex-matched controls who had uncomplicated malaria ( N = 44). Plasma concentrations of IgG against a detergentsoluble extract of P. falciparum schizonts were measured by quantitative ELISA, using indirect standardization. Among the Karitiana, the concentrations of anti-parasite antibodies of all subclasses increased with age, and there was no correlation between age and the proportion of such antibodies which was cytophilic. The predominance of cytophilic IgG1 and non-cytophilic IgG2 antibodies in all age-groups of the Karitiana provides an example of an intermediate pattern of immune responses to P. falciparum which contrasts with those previously described in both clinically immune and non-immune populations. Although mean concentrations of cytophilic IgG1 against P. falciparum were significantly higher in the controls than in the patients with severe malaria, there were no significant differences in other IgG subclasses. Lack of exposure to malaria in the past was associated with disease severity (odds ratio = 4.75; 95% confidence interval= 1.31-17.42), and may explain, at least partially, the occurrence of defective, low-IgG1 antibody responses to P. falciparum in those subjects who had severe malaria. [ABSTRACT FROM AUTHOR]

A case-control study to identify the risk factors for toxoplasmic encephalitis (TE) among HIV-infected patients with latent Toxoplasma gondii infection was performed in a teaching hospital in south-eastern Brazil. Although the subjects were all positive for serum IgG antibodies to Toxoplasma, some (the cases) developed TE during routine follow-up at the hospital whereas others (the controls) did not. Adjusted odds ratios (aOR) were estimated by multiple logistic regression after controlling for potential confounders. Only 46 (22%) of the 210 cases but 93 (45%) of the 205 controls were on prophylactic regimens with co-trimoxazole [aOR = 0.30; 95% confidence interval (CI) = 0.15-0.60]. Subjects with fewer than 100 (aOR = 37.09; CI =7.49-183.67) or between 100 and 200 CD4 cells/μl (aOR = 10.20; CI =2.00-51.90) were at substantially increased risk of developing TE than those with >400 CD4 cells/μl. Although the results of preliminary, unadjusted data analysis indicated that male sex and homosexual or bisexual activity might be additional risk factors, these associations were not found to be statistically significant by multiple regression analysis. In conclusion, no risk factors for TE other than low CD4 cell counts and failure to receive prophylaxis were found among HIV-infected Brazilian patients with past exposure to Toxoplasma. Seropositive patients with CD4 cell counts above 100/μl (the point at which specific prophylaxis is usually recommended) but below 200/μl might also benefit from effective anti-TE prophylaxis. [ABSTRACT FROM AUTHOR]

The polymorphic merozoite surface protein-2 (MSP-2) of Plasmodium falciparum is a major malaria-vaccine candidate. In the present study, PCR and hybridization with allelic-specific probes were used to type the Msp-2 gene from isolates from hypo-endemic Brazil (N = 113), meso-endemic Vietnam (N = 208) and holo-endemic Tanzania (N = 67). The typing methods were designed to group isolates into the dimorphic allelic families FC27 and IC1 and to detect possible between-family recombination events. The analysis was complemented by a comparison of 156 Msp-2 sequences from the GenBank database with 12 additional sequences obtained during the present study. Statistically significant differences were detected in pair-wise comparisons of the distribution of Msp-2 allelic types in Brazil and Vietnam, and in Brazil and Tanzania, but not in Vietnam and Tanzania. The extent of allelic diversity in the Msp-2 gene, as estimated by the total number of different alleles found in a given parasite population and the mean multiplicity of infections, clearly paralleled the levels of malaria endemicity in the study areas. However, no correlation between age and multiplicity of infections was found in the subjects. The patterns of Msp-2 diversity in Brazil appeared to be temporally stable, since no significant difference was observed in the distribution of Msp-2 allelic types among isolates collected, 10-13 years apart, in the same area of Rondônia. Despite the extensive sequence diversity found in Msp-2 alleles, especially in the central repetitive region of the molecule, several instances of identical or nearly identical alleles were found among isolates from different countries and regions, possibly as a result of extensive homoplasy. No recombinant allele was detected by molecular typing in any of the study sites, and the GenBank database included only 12 recombinant sequences (representing 7% of all reported Msp-2 sequences), all of them with an IC1-type 5' end and an FC27-type 3' end. A single, putative, crossover site was characterised for all recombinant alleles. Most of the allelic diversity observed was therefore attributable to variation in the repetitive region of the gene, instead of recombination between alleles of dimorphic families (as commonly found, for example, in the Msp-1 gene). The implications of these findings for studies on the genetic and antigenic diversity of malarial parasites are discussed. [ABSTRACT FROM AUTHOR]

The polymorphic, merozoite surface protein-1 (MSP-1) of Plasmodium falciparum, an antigen of the parasite's asexual blood-stages, is a major malaria-vaccine candidate. Nucleotide sequences of each variable domain or block of this antigen may be grouped into one of three possible allelic types (K1, MAD20 and RO33), and 24 major types of the msp-1 gene may be defined, as unique combinations of allelic types in these variable blocks. Isolates collected from the Brazilian Amazon, over a period of 14 years, have now been investigated, by PCR-based typing of the msp-1 gene. Thirteen of the 24 possible gene-types were identified, and 336 P. falciparum clones were fully typed among 239 isolates. Most parasites (87%) belonged to one of the seven most frequent gene-types. Marked temporal variation in the distribution of msp-1 variants was found when comparing parasites sampled in the same sites at intervals of at least 5 years. Spatial variations were also found when comparing parasites from both neighbouring and distant sites within the Amazon Basin. The between-population variance in the frequwncies of msp-1 allelic types found in Brazil, as estimated by Wright's FST statistic, is of similar magnitude to that found in previous world-wide comparisons. The potential implications of these findings for the development of an MSP-1-based, multivalent malaria vaccine are dicussed. [ABSTRACT FROM AUTHOR]

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.

The prevalences of intestinal parasitic infections were investigated, between 1995 and 1996, in a household-based sample of 1044 children aged <5 years who lived in the city of São Paulo, Brazil. Only 10.7% of the children were infected, the most prevalent parasites being Giardia duodenalis (5.5%), Ascaris lumbricoides (4.4%) and Trichuris trichiura (1.0%). A comparison between these data and results from two previous population-based surveys, completed in São Paulo in 1974 and 1985, revealed a dramatic decrease in the prevalence of intestinal helminths in this age-group, with less marked changes in the prevalence of Giardia, over the two past decades. Despite the low prevalence of malnutrition (2.4% of stunting and 0.6% of wasting) and intestinal parasites in this population, there was a significant association (P=0.05, after controlling for potential confounding variables) between helminth (but not Giardia) infection and height. The helminth-infected children had a mean height-for-age z-score of-0.412 [95% confidence interval (CI)=-0.637--0.186], compared with one of 0.015 (CI=-0.049-0.079) for the non-infected children. No significant relationship between intestinal parasitic infection and children's weight was detected. In conclusion, a small but significant negative relationship between intestinal helminthic infections and children's growth was detected in an urban environment with low prevalences of both intestinal parasitic infection and malnutrition.

We have compared Duffy blood group genotype distribution, as determined by polymerase chain reaction with allele-specific primers, in 68 Plasmodium vivax-infected patients and 59 non-vivax malaria controls from Rondônia, Brazil. Homozygosity for the allele Fy, which abolishes Duffy antigen expression on erythrocytes, was observed in 12% non-vivax controls but in no P. vivax patient. However, no significant association was found between Fy heterozygosity and protection against P. vivax. The Fy x allele, which has recently been associated with very weak erythrocyte expression of Duffy antigen, was not found in local P. vivax patients.

The merozoite surface protein-2 (MSP-2) is a major vaccine candidate for the asexual blood stage of Plasmodium falciparum. MSP-2 is essentially dimorphic, and allelic families are named after the representative isolates FC27 and IC1. The polymorphic central region contains immunodominant repeats, which vary in number, length, and sequence within and between allelic families. We have examined the antibody recognition of repeat regions from both MSP-2 allelic families expressed as recombinant fusion peptides. The results are summarized as follows. (1) Immunization of mice with the fusion peptides elicited IgG antibodies that cross-reacted with the native MSP-2 molecule in an allelic family-specific manner. (2) These mouse antibodies recognized the recombinant proteins in both a variant-specific and a family-specific manner, as shown in inhibition immunoassays. Antibodies raised against the peptide FC27 seemed to be essentially variant-specific, since the soluble form of the S20 antigen (a member of FC27 family) had relatively little inhibitory effect on them. (3) The overall pattern of human IgG antibody responses to MSP-2 in Karitiana Indians, a population continuously exposed to hypoendemic malaria in the Brazilian Amazon Region, differs from that described in hyperendemic areas in Africa and Papua New Guinea in two important features: there was no clear age-dependent increase in the prevalence and mean concentration of specific IgG antibodies, and there was no skewing towards the IgG3 subclass in antibody responses. (4) The relatively poor correlation between concentrations of IgG antibodies that are specific for members of the same allelic family suggests that recognition of MSP-2 peptides by naturally acquired antibodies was largely variant-specific in this population. The potential role of naturally acquired variant-specific antibodies in immune evasion, by selecting mutant parasites carrying insertions or deletions of repeat sequences, is briefly discussed.

Two polymerase chain reaction (PCR) protocols showed low sensitivity (36% and 53% for TB AMPLICOR and MPB64 nested PCR, respectively), when compared with classic microbiological methods (73% and 54% for Ziehl-Neelsen staining and culture, respectively), in the diagnosis of tuberculous meningitis in 91 patients in southeastern Brazil. Only three PCR-positive, microbiologically negative patients were found. Analysis of sequential cerebrospinal fluid samples by nested PCR detected Mycobacterium tuberculosis DNA up to 29 days after the introduction of antituberculosis chemotherapy.

The merozoite surface protein-1 (MSP-1) is a major vaccine candidate for the asexual blood stage of malaria. We examined both the extent of sequence diversity in block 17, the 3' end of Msp-1 gene coding for a 19-kDa polypeptide (MSP-1(19)) putatively involved in red blood cell binding, and the patterns of linkage disequilibrium between polymorphic sites throughout the Msp-1 locus. The parasite population sample consisted of Plasmodium falciparum isolates collected between 1985 and 1998 in Rondĵnia, an area of hypoendemic malaria transmission in the southwestern Brazilian Amazon. Results were summarized as follows. (1) Seven block-17 sequence variants or haplotypes were found among 130 isolates, including two new haplotypes (novel combinations of previously reported amino acid replacements), here named Brazil-1 (E-TSR-F) and Brazil-2 (Q-TSR-F). (2) As previously shown for other Msp-1 polymorphisms, frequencies of block-17 haplotypes displayed significant temporal variation. (3) Extensive linkage disequilibrium was demonstrated between neighboring dimorphic sites within block 17, as well as between polymorphisms at the 5' and 3' ends of Msp-1 (map distance range: 3.83-4.99 kb). (4) The overall patterns of linkage disequilibrium within Msp-1 remained stable over a period of nearly one decade, and examples of possible 'epidemic' expansion of parasites carrying particular Msp-1 alleles were found in the 1980s and 1990s. These results are discussed in relation to the population biology of P. falciparum and the development of malaria vaccines based on MSP-1.

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.

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.

Field isolates of Plasmodium falciparum collected from endemic areas of Southeast Asia, Solomon Islands, tropical African countries and Brazil were analyzed for the genetic diversity of the exon II of serine repeat antigen gene (SERA) by sequencing of genomic DNA. Of sixty-nine isolates, as compared to the reported FCR3, K1 and Honduras-1 types of exon II sequences, 5, 9 and 20 new allelic forms were found in 23 isolates of the FCR3 type, 36 of the K1 type and 10 of the Honduras-1 type. A group of novel non-synonymous substitutions, 4 new insertions and 3 new deletions of octamer units were found in the octamer repeat region (OR) of the exon II, and most of them clustered within a 40-residues domain. An octamer "SNPVSSEP" revealed in the OR was confirmed as a new repeat unit. Based on the sequences of the serine repeat region (SR) of the exon II, the allelic forms of the Honduras-1 type were conjectured to be the recombinant forms between the K1 type and FCR3 type. The allelic forms of K1 type with less or more repeat serine residues in the serine stretch of the SR than the reported 21 serine residues had most of the variations in the OR. Moreover, a biased geographical distribution of allelic forms was observed. Isolates from African and Southeast Asian countries accounted for most of the new allelic forms (29/33). All of the three types were detected in Southeast Asia but none of the FCR3 type in Africa. One of two groups of FCR3 new allelic forms was found solely in Brazil while another was mainly in Solomon Islands.

Multilocus genotyping of microbial pathogens has revealed a range of population structures, with some bacteria showing extensive recombination and others showing almost complete clonality. The population structure of the protozoan parasite Plasmodium falciparum has been harder to evaluate, since most studies have used a limited number of antigen-encoding loci that are known to be under strong selection. We describe length variation at 12 microsatellite loci in 465 infections collected from 9 locations worldwide. These data reveal dramatic differences in parasite population structure in different locations. Strong linkage disequilibrium (LD) was observed in six of nine populations. Significant LD occurred in all locations with prevalence <1% and in only two of five of the populations from regions with higher transmission intensities. Where present, LD results largely from the presence of identical multilocus genotypes within populations, suggesting high levels of self-fertilization in populations with low levels of transmission. We also observed dramatic variation in diversity and geographical differentiation in different regions. Mean heterozygosities in South American countries (0.3-0.4) were less than half those observed in African locations (0. 76-0.8), with intermediate heterozygosities in the Southeast Asia/Pacific samples (0.51-0.65). Furthermore, variation was distributed among locations in South America (F:(ST) = 0.364) and within locations in Africa (F:(ST) = 0.007). The intraspecific patterns of diversity and genetic differentiation observed in P. falciparum are strikingly similar to those seen in interspecific comparisons of plants and animals with differing levels of outcrossing, suggesting that similar processes may be involved. The differences observed may also reflect the recent colonization of non-African populations from an African source, and the relative influences of epidemiology and population history are difficult to disentangle. These data reveal a range of population structures within a single pathogen species and suggest intimate links between patterns of epidemiology and genetic structure in this organism.

Most studies of genetic variability of Plasmodium falciparum have focused on protein antigens and the genes that encode them. The consensus is that populations exhibit high levels of genetic polymorphism, most notably the genes encoding surface proteins of the merozoite (Msp1, Msp2) and the sporozoite (Csp). The age and derivation of this variation is a subject that warrants further careful consideration, as discussed here by Stephen Rich, Marcelo Ferreira and Francisco Ayala.

We have compared results of Plasmodium species identification obtained with conventional on-site microscopy of Giemsa-stained thick smears (GTS) and a semi-nested polymerase chain reaction (PCR) in 96 malaria patients from Rondônia, Western Brazilian Amazon. Mixed-species infections were detected by PCR in 30% patients, but no such case had been found on GTS. Moreover, P. malariae infections were detected in 9 of 96 patients (10%) by PCR, but were not identified by local microscopists. The potential impact of species misidentification on malaria treatment and control is discussed.

Objective: To show how a mathematical model can be used to describe and to understand the malaria transmission., Methods: The effects on malaria transmission due to the impact of the global temperature changes and prevailing social and economic conditions in a community were assessed based on a previously presented compartmental model, which describes the overall transmission of malaria., Results/conclusions: The assessments were made from the scenarios produced by the model both in steady state and dynamic analyses. Depending on the risk level of malaria, the effects on malaria transmission can be predicted by the temperature ambient or local social and-economic conditions.

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.

The polymorphic merozoite surface protein (MSP-1) of Plasmodium falciparum is a major asexual blood-stage malaria vaccine candidate. The impact of allelic diversity on recognition of MSP-1 during the immune response remains to be investigated in areas of hypoendemicity such as the Brazilian Amazon region. In this study, PCR was used to type variable regions, blocks 2, 4, and 10, of the msp-1 gene and to characterize major gene types (unique combinations of allelic types in variable blocks) in P. falciparum isolates collected across the Amazon basin over a period of 12 years. Twelve of the 24 possible gene types were found among 181 isolates, and 68 (38%) of them had more than one gene type. Temporal, but not spatial, variation was found in the distribution of MSP-1 gene types in the Amazon. Interestingly, some gene types occurred more frequently than expected from random assortment of allelic types in different blocks, as previously found in other areas of endemicity. We also compared the antibody recognition of polymorphic (block 2), dimorphic (block 6), and conserved (block 3) regions of MSP-1 in Amazonian malaria patients and clinically immune Africans, using a panel of recombinant peptides. Results were summarized as follows. (i) All blocks were targeted by naturally acquired cytophilic antibodies of the subclasses IgG1 and IgG3, but the balance between IgG1 and IgG3 depended on the subjects' cumulative exposure to malaria. (ii) The balance between IgG1 and IgG3 subclasses and the duration of antibody responses differed in relation to distinct MSP-1 peptides. (iii) Antibody responses to variable blocks 2 and 6 were predominantly type specific, but variant-specific antibodies that target isolate-specific repetitive motifs within block 2 were more frequent in Amazonian patients than in previously studied African populations.

Plasmodium ovale and Plasmodium malariae, two of the four human malaria parasites, are usually found at very low prevalence in East Asia, even in areas with intense malaria transmission. In this article, Fumihiko Kawamoto, Qing Liu, Marcelo Ferreira and Indah Tantular review data obtained in recent field surveys, using alternative diagnostic methods such as acridine orange staining and PCR-based methods, to evaluate the prevalence of these two malaria species in East Asia. They argue that these species might be much more prevalent in East Asia than reported previously. In addition, they discuss the implications of sequence variations found in the small subunit ribosomal RNA genes of the two species targeted by diagnostic PCR and compare morphological criteria for speciation of malaria parasites stained with Giemsa and acridine orange.

Background: The 19 kDa C-terminal region of the merozoite surface protein 1 (MSP1(19)) has been suggested as candidate for part of a subunit vaccine against malaria. A major concern in vaccine development is the polymorphism observed in different plasmodial strains. The present study examined the extension and immunological relevance of the allelic polymorphism of the MSP1(19) from Plasmodium vivax, a major human malaria parasite., Materials and Methods: We cloned and sequenced 88 gene fragments representing the MSP1(19) from 28 Brazilian isolates of P. vivax. Subsequently, we evaluated the reactivity of rabbit polyclonal antibodies, a monoclonal antibody, and a panel of 80 human sera to bacterial and yeast recombinant proteins representing the two allelic forms of P. vivax MSP1(19) described thus far., Results: We observed that DNA sequences encoding MSP1(19) were not as variable as the equivalent region of other species of Plasmodium, being conserved among Brazilian isolates of P. vivax. Also, we found that antibodies are directed mainly to conserved epitopes present in both allelic forms of the protein., Conclusions: Our findings suggest that the use of MSP1(19) as part of a subunit vaccine against P. vivax might be greatly facilitated by the limited genetic polymorphism and predominant recognition of conserved epitopes by antibodies.

We have investigated the seroprevalence of hepatitis B and C among Karitiana Indians (n = 119) living in the State of Rondônia, southwestern Brazilian Amazon. The prevalences of anti-HBs and anti-HBc were 16.1% and 35.3%, respectively, with HBsAg being found in only four (3.4%) subjects. Anti-HCV antibodies were detected in two subjects (1.7%). Age-stratified prevalence data suggest that both vertical and horizontal (the last among adults) routes of HBV transmission are important in this community.

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.

The merozoite surface protein-1 (MSP-1) locus of Plasmodium falciparum codes for a major asexual blood-stage antigen currently proposed as a major malaria vaccine candidate. The protein, however, shows extensive polymorphism, which may compromise its use in sub-unit vaccines. Here we compare the patterns of allelic diversity at the MSP-1 locus in wild isolates from three epidemiologically distinct malaria-endemic areas: the hypoendemic southwestern Brazilian Amazon (n = 54), the mesoendemic southern Vietnam (n = 238) and the holoendemic northern Tanzania (n = 79). Fragments of the variable blocks 2, 4a, 4b and 6 or 10 of this single-copy gene were amplified by the polymerase chain reaction, and 24 MSP-1 gene types were defined as unique combinations of allelic types in each variable block. Ten different MSP-1 types were identified in Brazil, 23 in Vietnam and 13 in Tanzania. The proportion of genetically mixed infections (isolates with parasites carrying more than one MSP-1 version) ranged from 39% in Brazil to 44% in Vietnam and 60% in Tanzania. The vast majority (90%) of the typed parasite populations from Brazil and Tanzania belonged to the same seven most frequent MSP-1 gene types. In contrast, these seven gene types corresponded to only 61% of the typed parasite populations from Vietnam. Non-random associations were found between allelic types in blocks 4a and 6 among Vietnamese isolates, the same pattern being observed in independent studies performed in 1994, 1995 and 1996. These results suggest that MSP-1 is under selective pressure in the local parasite population. Nevertheless, the finding that similar MSP-1 type frequencies were found in 1994 and 1996 argues against the prominence of short-term frequency-dependent immune selection of MSP-1 polymorphisms. Non-random associations between MSP-1 allelic types, however, were not detected among isolates from Brazil and Tanzania. A preliminary analysis of the distribution of MSP-1 gene types per host among isolates from Tanzania, but not among those from Brazil and Vietnam, shows significant deviation from that expected under the null hypothesis of independent distribution of parasites carrying different gene types in the human hosts. Some epidemiological consequences of these findings are discussed.

The extent of allelic diversity at the Merozoite Surface Protein-1 locus of Plasmodium falciparum (PfMSP-1) was examined in isolates collected from symptomatic patients living in a mesoendemic area in southern Vietnam. The variable blocks 2, 4 and 10 were typed by polymerase chain reaction and 24 PfMSP-1 gene types were defined as unique combinations of allelic types detected in each variable block. Nineteen PfMSP-1 gene types were identified and 182 parasite populations were fully typed among 102 isolates. Forty-eight (47%) patients harbored more than one typed parasite population, and one patient had at least eight genetically distinct subpopulations. As previously shown in the same endemic area, recombination between blocks 4 and 10 was significantly less frequent than expected from random assortment of allelic types. The distribution of PfMSP-1 gene types, however, did not differ significantly from that observed in isolates collected in the same area 17-24 mo before the present study. Furthermore, the prevalence of the most common gene types and the average number of different gene types harbored by the same host did not decrease with age. This argues against the prominence of frequency-dependent immune selection of PfMSP-1 polymorphisms in this parasite population.

Allelic variation in the Plasmodium falciparum merozoite surface protein 1 (MSP1) gene is expressed as an association of allelic types in variable blocks. In this study, a PCR strategy that can detect 24 different MSP1 association types was used to investigate allelic variation in the MSP1 gene. We identified 236 distinct association type clones in 136 wild isolates collected from southern Vietnam, analysis of which revealed that (1) recombination between two representative allelic types in the central part of the MSP1 gene did not exist, (2) frequency distribution of MSP1 association types did not differ in different population groups, and (3) particular MSP1 association types were predominant. Statistical analysis for the association of allelic types indicated significant, nonrandom associations between blocks 4 and 6 but not between blocks 2 and 4, and 2 and 6. These results suggest that selection operates in favor of particular MSP1 association types. In addition, direct sequencing of 31 isolates confirmed reported sequence substitutions in the C-terminal 19-kDa Cys-rich region of MSP1, supporting a notion of limited variations in this region, a strong vaccine candidate molecule.

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.

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.

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.

The hypotheses that iron-deficient hosts are less susceptible to severe malaria and that iron supplementation aggravates infection have been supported by some clinical and experimental evidence. In the present study, the course of Plasmodium berghei infection was monitored in an experimental model of dietary iron deficiency and iron supplementation. Weanling Wistar rats were fed purified diets with different iron concentrations: 20 mg/kg (Group D, n = 24), 50 mg/kg (Group N, n = 24) and 100 mg/kg (Group S, n = 12). After 15 d, rats from Group D were anemic (mean hemoglobin 81 g/l). The next day, 12 rats from Group D (thereafter Group DS) and 12 rats from Group N (thereafter Group NS) were transferred to the same iron-supplemented diet as in Group S, whereas the remaining animals (Groups D, N and S) were maintained on the original diets for further 14 d. At that time, 9 rats from each group were inoculated intraperitoneally with 10(6) erythrocytic parasites (P. berghei ANKA strain), whereas 3 rats from each group remained as noninfected controls. All animals were killed 14 d after inoculation, when significantly lower levels of hemoglobin, serum iron and percent transferrin saturation were found in infected animals from Group D compared with all other groups. However, the time course of parasitemias was similar in all groups. These data indicate that the development of P. berghei was neither suppressed by iron deficiency nor enhanced by iron supplementation in this model. Furthermore, iron repletion during infection did produce a noticeable improvement of hematological variables in previously iron-deficient animals.

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.

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.

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.

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.

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.