Polymorphism in the circumsporozoite protein of the human malaria parasite Plasmodium vivax.

The circumsporozoite (CS) protein that covers the surface of infectious sporozoites is a candidate antigen in malaria vaccine development. To determine the extent of B- and T-epitope polymorphism and to understand the mechanisms of antigenic variability, we have characterized the CS protein gene of Plasmodium vivax from field isolates representing geographically distant regions of Papua New Guinea (PNG) and Brazil. In the central repeat region of the CS protein, in addition to variation in the number of repeats, an array of mutations was observed which suggests that point mutations have led to the emergence of the variant CS repeat sequence ANGA(G/D)(N/D)QPG from GDRA(D/A)GQPA. Outside the repeat region of the protein, the nonsilent nucleotide substitutions of independent origin are localized in three domains of the protein that either harbor known T-cell determinants or are analogous to the Plasmodium falciparum immunodominant determinants, Th2R and Th3R. We have found that, with the exception of one CS clone sequence that was shared by one P. vivax isolate each from PNG and Brazil, the P. vivax CS protein types can be grouped into Papuan and Brazilian types. These results suggest that an in-depth study of parasite population dynamics is required before field trials for vaccine formulation based on polymorphic immunodominant determinants are conducted.

Extreme geographical fixation of variation in the Plasmodium falciparum gamete surface protein gene Pfs48/45 compared with microsatellite loci.

Comparing patterns of genetic variation at multiple loci in the genome of a species can potentially identify loci which are under selection. The large number of polymorphic microsatellites in the malaria parasite Plasmodium falciparum are available markers to screen for selectively important loci. The Pfs48/45 gene on Chromosome 13 encodes an antigenic protein located on the surface of parasite gametes, which is a candidate for a transmission blocking vaccine. Here, genotypic data from 255 P. falciparum isolates are presented, which show that alleles and haplotypes of five single nucleotide polymorphisms (SNPs) in the Pfs48/45 gene are exceptionally skewed in frequency among different P. falciparum populations, compared with alleles at 11 microsatellite loci sampled widely from the parasite genome. Fixation indices measuring inter-population variance in allele frequencies (F(ST)) were in the order of four to seven times higher for Pfs48/45 than for the microsatellites, whether considered (i) among populations within Africa, or (ii) among different continents. Differing mutational processes at microsatellite and SNP loci could generally affect the population structure at these different types of loci, to an unknown extent which deserves further investigation. The highly contrasting population structure may also suggest divergent selection on the amino acid sequence of Pfs48/45 in different populations, which plausibly indicates a role for the protein in determining gamete recognition and compatibility.

Origin of Plasmodium falciparum malaria is traced by mitochondrial DNA.

The origin and geographical spread of Plasmodium falciparum is here determined by analysis of mitochondrial DNA sequence polymorphism and divergence from its most closely related species P. reichenowi (a rare parasite of chimpanzees). The complete 6 kb mitochondrial genome was sequenced from the single known isolate of P. reichenowi and from four different cultured isolates of P. falciparum, and aligned with the two previously derived P. falciparum sequences. The extremely low synonymous nucleotide polymorphism in P. falciparum (pi=0.0004) contrasts with the divergence at such sites between the two species (kappa=0.1201), and supports a hypothesis that P. falciparum has recently emerged from a single ancestral population. To survey the geographical distribution of mitochondrial haplotypes in P. falciparum, 104 isolates from several endemic areas were typed for each of the identified single nucleotide polymorphisms. The haplotypes show a radiation out of Africa, with unique types in Southeast Asia and South America being related to African types by single nucleotide changes. This indicates that P. falciparum originated in Africa and colonised Southeast Asia and South America separately.

Diversity in the immunodominant determinants of the circumsporozoite protein of Plasmodium falciparum parasites from malaria-endemic regions of Papua New Guinea and Brazil.

To determine the nature and extent of variation in the T cell sites of the Plasmodium falciparum circumsporozoite (CS) protein, a candidate antigen in the development of a malaria vaccine, we cloned and sequenced 69 recombinant clones of the CS protein gene representing 18 and 17 P. falciparum isolates from infected individuals from Madang, Papua New Guinea (PNG), a holoendemic malaria region, and Paragaminos and Jacunda, Brazil, relatively low endemic regions, respectively. As previously known, the amino acid sequence polymorphism was restricted to the three immunodominant regions of the protein, Th1R-N1, Th2R, and Th3R. While some of the observed nonsilent mutations in the T cell determinants of the CS protein were similar to those previously identified, we have found new amino acid changes in each of the polymorphic sequences in parasites from PNG and Brazil. A comparison of the CS epitope sequences of parasites from PNG and Brazil with the previously known CS epitope sequences of parasites from Brazil and The Gambia showed the following: 1) polymorphism was found in the Th1R-N1, Th2R, and Th3R region; however, while amino acid substitutions in the Th1R-N1 and Th2R region tended to be conservative, the substitutions found in the Th3R region were not, suggesting that the Th3R epitope may be rapidly evolving to allow parasites to escape host antiparasite cytotoxic T cell-enforced immune responses, and 2) the CS proteins of P. falciparum from high malaria-transmission regions (PNG and The Gambia) appear more polymorphic than the CS proteins of parasites from relatively low malaria-endemic regions in Brazil, where P. falciparum infection has been recently established.

Vertebrate hosts and vectors of Trypanosoma rangeli in the Amazon Basin of Brazil.

A total of 46 Trypanosoma rangeli stocks were isolated from naturally infected mammals and triatomine vectors. Twenty-two stocks were from the common opossum (Didelphis marsupialis), one from the brown "4-eyed" opossum (Metachirus nudicaudatus), one from the anteater (Tamandua tetradactyla), one from the coati (Nasua nasua), seven from Rhodnius pictipes and 14 from Rhodnius robustus. Two stocks were also isolated from recently fed sandflies (Lutzomyia sp., Shannoni group). The stocks were identified as T. rangeli on the basis of natural or experimental salivary gland infections in Rhodnius, inoculative (anterior station) transmission to mice, morphological parameters in parasitemic mice and comparisons of isozyme profiles with a known stock of T. rangeli isolated from man. Three other trypanosome stocks from D. marsupialis, T. tetradactyla and the three-toed sloth (Bradypus tridactylus) were morphologically similar to T. rangeli in culture but had quite different isozyme profiles and were not identified. It is concluded that T. rangeli is widely distributed in Amazonas, Pará and Rondonia States of Brazil, and probably extends into other regions where R. pictipes and R. robustus are known to occur. R. pictipes is light-attracted into houses and occasionally transmits Chagas' disease to man. It is likely that T. rangeli is also occasionally transmitted to man in the Amazon basin.

Flagellate infections of Brazilian sand flies (Diptera: Psychodidae): isolation in vitro and biochemical identification of Endotrypanum and Leishmania.

Flagellate infections were found in 1,063 of 18,895 sand flies collected in the states of Amazonas, Pará, Rondonia and Acre, Brazil. Infection rates were 13.4% (species group Shannoni); 7.5% (subgenus Nyssomyia); 6.7% (subgenus Lutzomyia series Cruciata); 0.5% (genus Psychodopygus) and 3.1% for other sand flies (various subgenera). Leishmania braziliensis guyanensis and L. mexicana amazonensis were isolated, respectively, from the known vectors, Lutzomyia umbratilis and L. flaviscutellata. Single stocks of L. braziliensis-like and L. mexicana-like organisms were isolated, respectively, from L. whitmani and L. yuilli. Thirty-eight flagellate stocks, isolated by direct culture from sand flies were characterized in detail by morphology in culture, behavior in hamsters and mice and by enzyme profiles. Sixteen stocks from Lutzomyia sp. (Shannoni group) were identified as Endotrypanum schaudinni; 8 stocks from Lutzomyia sp. (Shannoni group) were identified as Endotrypanum sp.; 7 stocks from Psychodopygus ayrozai and P. paraensis were identified as Leishmania sp. previously isolated from the armadillo, Dasypus novemcinctus; 2 stocks of Trypanosoma rangeli were isolated from recently fed Lutzomyia sp. (Shannoni group) sand flies; the remaining 5 stocks from L. umbratilis and L. yuilli could not be identified. Observations suggested that Shannoni group sand flies were the natural vectors of Endotrypanum. Leishmania sp. infections in the man-biting flies P. ayrozai and P. paraensis were restricted to the midgut and associated with recent bloodmeals. Unidentified flagellates in L. umbratilis and L. yuilli were distributed throughout the digestive tract with no trace of bloodmeals.

Distribution of Plasmodium vivax variants (VK210, VK247 and P. vivax-like) in three endemic areas of the Amazon region of Brazil and their correlation with chloroquine treatment.

The present study evaluated the glass fibre membrane (GFM)-polymerase chain reaction (PCR)-enzyme-linked immunosorbent assay (ELISA) technique for genotyping the Plasmodium vivax variants, to verify the distribution of P. vivax variants (VK210, VK247 and P. vivax-like) in parts of Brazil and their correlation with levels of parasitaemia, previous malaria experience and clearance of parasitaemia linked to different treatment schedules. The samples were taken from individuals living in Macapá, Porto Velho and Belém, all of which are endemic areas of vivax malaria in the Amazon region of Brazil. Blood samples were collected on GFMs. The gene that codes for the circumsporozoite proteins of P. vivax variants was amplified by PCR and the amplified fragments were hybridized to variant-specific, digoxigenin-labelled oligonucleotide probes by ELISA. The GFM-PCR-ELISA technique was shown to be accurate for epidemiological surveys of the vivax complex. All variants were detected in all 3 areas, but only P. vivax VK210 was found as a single agent of infection, while the other 2 occurred as mixed infections. The P. vivax-like variant was found to be associated with low parasitaemia and VK210 with the highest parasitaemia levels; none of the P. vivax variants was linked with a previous malaria experience. In all cases parasitaemia clearance was identical regarding the type of treatment and consequently it is not possible to confirm the previously reported correlation between P. vivax genotype and response to chloroquine.

Leishmaniasis in Brazil: XVIII. Further evidence incriminating the fox Cerdocyon thous (L) as a reservoir of Amazonian visceral leishmaniasis.

Major endemic areas of visceral leishmaniasis in Brazil are located in the drier, poorly forested regions, principally in the northeastern States such as Ceará and Bahia. Cases of the human disease in the Amazon Region are rare, very sporadic, and seldom present opportunities for epidemiological study. Following the report of a fatal case near Salvaterra, the Island of Marajó, Pará State, a preliminary investigation has resulted in the isolation of a parasite regarded as Leishmania donovani chagasi from the viscera and skin of an apparently healthy fox, Cerdocyon thous, captured in the same locality. This represents the third recorded isolation of the parasite from this species of fox in the Amazon Region. The inapparent nature of the infections supports the suggestion that this canid may represent the primitive natural host of L. d. chagasi. C. thous is commonly associated with forested or wooded areas, and enzymic profiles for the enzymes ASAT, ALAT, PGM, GPI, MDH, MPI, G6PD, PEP and ACP failed to distinguish an isolate of L. d. chagasi from this animal in Pará from others obtained from cases of human visceral leishmaniasis in the neighbouring States of Maranhão, Ceará and Bahia. This suggests that the major, present-day endemics may have originated from a primary silvatic enzootic.

Isozyme heterogeneity and numerical taxonomy of Trypanosoma cruzi stocks from Chile.

Fifty-three stocks of Trypanosoma cruzi were isolated in Chile, 13 from patients, 32 from the domestic triatomine vector Triatoma infestans, and 8 from the silvatic and peridomestic vector T. spinolai. The majority of isolates from triatomine bugs were made by the direct culture of infected faeces. The 53 stocks and a single clone were characterized by a combination of starch-gel and cellulose acetate enzyme electrophoresis. Three groups of T. cruzi stocks were apparent from either simple visual comparisons of isozyme profiles or numerical taxonomy. The groups were designated Chilean zymodeme (Z) 1, which was similar to Brazilian Z1, Chilean Z2a, similar to Brazilian Z2 and Chilean Z2b, similar to Bolivian Z2 and with prominent heterozygous isozyme profiles. Chilean Z1 was isolated only from T. spinolai colonizing farm walls inhabited by the rodent Octodon degus. Chilean Z2a and Z2b were both isolated from domestic T. infestans T. infestans and man, in some cases within the same household. Hardy-Weinberg equilibria were not found amongst a group of 22 stocks from a single locality and deviations from theoretical Hardy-Weinberg distributions were compatible with the absence of genetic exchange in the sampled population of T. cruzi.

Some methods for the enzymic characterization of Latin-American Leishmania with particular reference to Leishmania mexicana amazonensis and subspecies of Leishmania hertigi.

30 Brazilian stocks of Leishmania mexicana amazonensis and 13 stocks of subspecies of Leishmania hertigi were characterized by starch-gel electrophoresis, using 18 enzymes selected from a total of 36 investigated. L. m. amazonensis was separable from subspecies of L. hertigi by enzymic profiles of 11 enzymes. The L. m. amazonensis stocks, which were from a wide range of hosts in a large geographical area, were enzymically extremely homogeneous, and could only be subdivided on two enzymes; sub-groups did not relate to each other or to any differences in epidemiological characters, including the clinical form of the human disease. 12 stocks regarded as L. hertigi deanei, that were isolated from Coendou prehensilis prehensilis and Coendou sp. in Pará State, Brazil, were separable into two sub-groups by three enzymes. A single stock of L. hertigi hertigi from Panama was separable from both enzymic sub-groups of L. h. deanei, in each case by three enzymes. The significance of these and other characters of diversity is discussed, together with the use of enzymes for the identification of the leishmaniae.

Chagas's disease in the Amazon Basin: Ii. The distribution of Trypanosoma cruzi zymodemes 1 and 3 in Pará State, north Brazil.

In Pará State, Brazil, 123 Trypanosoma cruzi stocks were isolated from 12 silvatic mammal species, five silvatic triatomine species and individuals with acute Chagas's disease. 100 T. cruzi stocks were identified as zymodeme (Z) 1, 17 as Z3 and 6 as Z3 with Z1 ASAT character, but none were T. cruzi Z2. Z1 was predominantly isolated from arboreal mammals, especially Didelphis marsupialis; Z3 was mainly found in terrestrial or burrowing mammals, particularly Dasypus novemcinctus and Monodelphis brevicaudata. It is not clear whether gene exchange occurs between the groups designated as zymodemes but the enzymic "distance" between T. cruzi Z1, Z2 and Z3, their different geographical distributions, host associations and local transmission cycles support the view that these zymodemes represent taxonomic units of fundamental epidemiological significance. T. cruzi (Z1) was isolated for the first time from the silky anteater (Cyclopes didactylus).

Trypanosoma cruzi: zymodemes associated with acute and chronic Chagas' disease in central Brazil.

The clinical characteristics of acute and chronic Chagas' disease in central Brazil are described (29 acute cases and 111 chronic cases). The geographical distribution of Trypanosoma cruzi zymodemes in this region was mapped. Zymodeme (Z) 1 was identified in 12 acute cases, Z2 in 13 and repeated xenodiagnosis gave the same zymodeme identification. The clinical pictures of the Z1 and Z2 acute phases were similar. Resistance to benznidazole treatment occurred after either Z1 or Z2 acute infections. Only 14 positive xenodiagnosis were obtained from the 111 chronic phase patients examined. For 12 of these 14 patients the zymodeme was identified. All 12 carried Z2, 10 of whom had mega involvement. There were several possible explanations for the failure to detect T. cruzi Z1 in chronic Chagas' disease with mega syndromes: suggestions were made for follow-up investigations.

Evaluation of the Amazon River delta as a barrier to gene flow for the regional malaria vector, Anopheles aquasalis (Diptera: Culicidae) in northeastern Brazil.

The Neotropical malaria vector Anopheles aquasalis Curry is distributed predominantly along the Atlantic and Pacific Coasts because of its tolerance for breeding in salt water. We tested the hypothesis that the freshwater Amazon River acts as a barrier to gene flow in northeastern Brazil, by examining variation at a 588-nucleotide fragment of the mitochondrial cytochrome oxidase Igene from five populations. We identified 15 haplotypes of which 5 were shared both (1) between sample localities and (2) across the Amazon River Delta. Sequence divergence ranged from 0.0017-0.0272 (average = 0.0137). Estimates of genetic subdivision based on the presence of the Amazon Riverwere greatest within localities (phi = 0.029) and among regions (phi = 0.018), followed by among localities (phi = 0.011), but none were significant. Parsimony, neighbor-joining, and Nested Clade Analyses were used to estimate relationships among populations and infer evolutionary processes. Two phylogenetically distinct clusters of populations were moderately supported by parsimony. Neighbor-joining trees were poorly resolved, thus providing no geographical resolution and no support for the Amazon River as a barrier to migration. Phylogeographic structure as detected by the Nested Clade Analysis was consistent with restricted gene flow coupled with isolation by distance. Taken together, these analyses suggest that the localities within this region of northeastern Brazil constitute a single large population of An. aquasalis that spans the Amazon Delta.

Simplified diagnosis of malaria infection: GFM/PCR/ELISA a simplified nucleic acid amplification technique by PCR/ELISA.

We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.

[Comparison of 4 techniques for the diagnosis of Giardia lamblia in stool of children from Belém city, Pará State, Brazil]. / Comparação de quatro métodos laboratoriais para diagnóstico da Giardia lamblia em fezes de crianças residentes em Belém, Pará

We report the evaluation of four techniques for Giardia lamblia diagnosis in children's stool. The Iron haematoxilin staining and direct examination with lugol showed lower positivity, while the method of Faust et al. Continues to be a good option for G. lamblia diagnosis and Immunoenzymatic assay increases the detection of this parasite.

[Diagnosis of intestinal amebiasis using coproscopic and immunological methods in a population sample in greater metropolitan Belém, Pará, Brazil]. / Diagnóstico de amebíase intestinal utilizando métodos coproscópicos e imunológicos em amostra da população da área metropolitana de Belém, Pará, Brasil.

We compare diagnostic methods for Entamoeba histolytica in fecal samples from the city of Belém, Pará, Brazil. We analyze stool samples from children and adults (Group I); stool and serum samples from adults (Group II); and stool samples from children (Group III). In groups I and III, we used direct examination with lugol (DM), Faust et al (FM), and ELISA (detection of E. histolytica anti-GIAP coproantigen) and in group II, DM, iron hematoxylin staining (IHS), FM, ELISA, and the indirect immunofluorescence test (IFAT) for detection of IgG antibodies. Positivity was 10.50% by DM plus FM and 28.99% by ELISA. There was no correlation between positivity and age group. In Group II (n = 87), the positive rate was 4.59% by DM plus FM, 8.04% by IHS, 4.59% by IFAT, and 21.83% by ELISA. The ELISA test was the most sensitive for all groups. IFAT alone is still not a useful tool for diagnosis of E. histolytica infection. The ELISA test is simple, performed in one-third of cases used for IHS and IFAT, and greatly improves quality of diagnosis. We recommend this as the method of choice for diagnosis of suspected E. histolytica infection.