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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. Key-words: Malaria diagnosis. PCR. Brazilian Amazon. Plasmodium malariae.Resumo Comparam-se neste trabalho resultados de identificação de espécie de Plasmodium obtidos com a microscopia convencional de gotas espessas coradas pelo Giemsa, realizada no local, e um protocolo semi-aninhado de reação em cadeia da polimerase (PCR) em 96 pacientes maláricos de Rondônia, Amazônia Ocidental Brasileira. Infecções envolvendo mais de uma espécie foram detectadas através de PCR em 30% dos pacientes, mas nenhum caso havia sido encontrado pelo exame de gotas espessas. Além disso, encontraram-se infecções por P. malariae por PCR em 9 dos 96 pacientes (10%), mas nenhuma havia sido encontrada pelos microscopistas locais. Discute-se o potencial impacto de erros de identificação de espécie sobre o tratamento e o controle da malária. Palavras-chaves: Diagnóstico da malária. PCR. Amazônia Brasileira. Plasmodium malariae.

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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.

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.

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.