Mariano Gustavo Zalis

pfcrt Polymorphism and the Spread of Chloroquine Resistance in Plasmodium falciparum Populations across the Amazon Basin

The widespread occurrence of drug-resistant malaria parasites in South America presents a formidable obstacle to disease control in this region. To characterize parasite populations and the chloroquine-resistance profile of Plasmodium falciparum in the Amazon Basin, we analyzed a DNA segment of the pfcrt gene, spanning codons 72-76, and genotyped 15 microsatellite (MS) markers in 98 isolates from 6 areas of Brazil, Peru, and Colombia where malaria is endemic. The K76T mutation, which is critical for chloroquine resistance, was found in all isolates. Five pfcrt haplotypes (S[tct]MNT, S[agt]MNT, CMNT, CMET, and CIET) were observed, including 1 previously found in Asian/African isolates. MS genotyping showed relatively homogeneous genetic backgrounds among the isolates, with an average of 3.8 alleles per marker. Isolates with identical 15-loci MS haplotypes were found in different locations, suggesting relatively free gene flow across the Amazon Basin. Allopatric isolates carrying SMNT and CMNT haplotypes have similar genetic backgrounds, although parasites carrying the CIET haplotype have some exclusive MS alleles, suggesting that parasites with CIET alleles were likely to have been introduced into Brazil from Asia or Africa. This study provides the first evidence of the Asian pfcrt allele in Brazil and a detailed analysis of P. falciparum populations, with respect to pfcrt haplotypes, in the Amazon Basin.

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase of isolates from the Amazon Region of Brazil

Since the late 1970s pyrimethamine-sulfadoxine (PS; FansidarTM Hoffman-LaRoche, Basel) has been used as first line therapy for uncomplicated malaria in the Amazon basin. Unfortunately, resistance has developed over the last ten years in many regions of the Amazon and PS is no longer recommended for use in Brazil. In vitro resistance to pyrimethamine and cycloguanil (the active metabolite of proguanil) is caused by specific point mutations in Plasmodium falciparum dihydrofolate reductase (DHFR), and in vitro resistance to sulfadoxine has been associated with mutations in dihydropteroate synthase (DHPS). In association with a proguanil-sulfamethoxazole clinical trial in Brazil, we performed a nested mutation-specific polymerase chain reaction to measure the prevalence of DHFR mutations at codons 50, 51, 59, 108 and 164 and DHPS mutations at codons 436, 437, 540, 581 and 613 at three sites in the Brazilian Amazon. Samples from two isolated towns showed a high degree of homogeneity, with the DHFR Arg-50/Ile-51/Asn-108 and DHPS Gly-437/Glu-540/Gly-581 mutant genotype accounting for all infections in Peixoto de Azevedo (n = 15) and 60% of infections in Apiacás (n = 10), State of Mato Grosso. The remaining infections in Apiacás differed from this predominant genotype only by the addition of the Bolivia repeat at codon 30 and the Leu-164 mutation in DHFR. By contrast, 17 samples from Porto Velho, capital city of the State of Rondônia, with much in- and out-migration, showed a wide variety of DHFR and DHPS genotypes.

Invasion Profiles of Brazilian Field Isolates of Plasmodium falciparum: Phenotypic and Genotypic Analyses

ABSTRACT The invasion of red blood cells (RBCs) by Plasmodium falciparum is dependent on multiple molecular interactions between erythrocyte receptors and parasite ligands. Invasion studies using culture-adapted parasite strains have indicated significant receptor heterogeneity. It is not known whether this heterogeneity reflects the parasite invasion arsenal in the field. We have studied the invasion phenotypes of 14 distinct field isolates from the Legal Amazon areas of Brazil by using erythrocyte invasion assays to investigate invasion into normal, enzyme-treated, and clinical-mutant RBCs. Analysis of these isolates revealed four distinct invasion profiles. Using En(a−) cells to get an unequivocal estimate of the use of glycophorin A (GPA) as a receptor, we found that the 175-kDa erythrocyte-binding antigen (EBA-175)/GPA pathway was used by a minority of the parasite isolates studied. Although polymorphism of region II domains at specific amino acid positions in both EBA-140 and EBA-181 was found in these field isolates, this did not correlate with invasion profiles and thus receptor selectivity. These studies have further confirmed the existence of a significant diversity of invasion pathways in nature and suggest that additional parasite ligands will have to be targeted to devise global vaccines that will work in the field.

Characterization of the pfmdr2 gene for Plasmodium falciparum

We report the characterization of the pfmdr2 gene which is a gene related to the P-glycoprotein family but with a somewhat different structure than the mdr genes. Based on DNA sequence analysis of genomic clones, we have discovered that the pfmdr2 gene has 10 predicted transmembrane domains and a single ATP-binding site. In a homology search using GenBank sequences, we discovered that the pfmdr2 gene has a significant homology with the hmt1 gene in yeast. The yeast hmt1 gene is involved in cadmium resistance and is hypothesized to transport cadmium containing complexes from the cell. We have further characterized the pfmdr2 gene expression by northern analysis and discovered that it is expressed in a stage-specific manner, only at the trophozoite stage and not in ring stages. We have prepared a rabbit antibody to a recombinant fusion protein expressing a portion of the pfmdr2 coding region. In IFA analysis, this antibody stains trophozoites and not ring stages. Western analysis reveals a protein of approximately 110 kDa which is consistent with the size of the predicted open reading frame based on DNA sequence analysis. Based on this analysis and previous work, there is no evidence for a change in pfmdr2 expression in drug-resistant versus drug-sensitive parasites.

Review of the etiologies of viral meningitis and encephalitis in a dengue endemic region.

OBJECTIVES To evaluate the etiology of viral meningitis and encephalitis in adults and adolescents living in areas affected by dengue. METHODS Over two years, adults and adolescents with diagnoses of viral encephalitis or meningitis were selected for study in Brazil. PCRs for dengue, enterovirus, HSV1 and 2 and cytomegalovirus were performed in CSF samples. Serum and CSF samples were tested for the presence of anti-dengue IgM antibodies. RESULTS The etiologies of encephalitis and meningitis were determined in 70% of cases (30/47). Dengue was the leading cause of encephalitis (47%) with normal CSF cellularity in 75% of these patients. HSV1 was found in 17.6% of the cases, two of which had mild encephalitis. Enterovirus was the most common cause of meningitis (50%), followed by HSV1 (15%), cytomegalovirus and dengue (10%, each). CONCLUSIONS We identified the viral agents causing encephalitis and meningitis in a higher proportion of cases than has been reported in other studies. Dengue was the most frequent cause of encephalitis, which surpassed HSV. In endemic areas, dengue should be investigated as an important cause of encephalitis. Normal CSF cellularity should not exclude dengue encephalitis. Enterovirus is known to be the leading cause of meningitis in children, but here we found it was also the main cause of the disease in adults. HSV1 should be investigated in patients with mild forms of encephalitis and meningitis.

Malaria diagnosis: standardization of a polymerase chain reaction for the detection of Plasmodium falciparum parasites in individuals with low-grade parasitemia.

Abstract In Brazil, no study has been done concerning the detection of malaria parasites by polymerase chain reaction (PCR) related to the diagnosis of Plasmodium falciparum malaria. In the present report we describe a highly sensitive methodology for malaria diagnosis using a nested PCR method based on amplification of the p126 P. falciparum gene detected by simple ethidium bromide staining. The P. falciparum Palo Alto strain (culture samples) was serially diluted in blood from an uninfected donor to a final level of parasitemia corresponding to 10–8% and was processed for PCR amplification. In each of these dilutions a parasitological examination was performed to compare the sensitivity with that of PCR amplification. Blood samples (field samples) were obtained from 51 malarious patients with positive thick blood smears (TBS) who were living in endemic regions of the Brazilian Amazon. They corresponded to 42 P. falciparum and 9 P. vivax cases, with parasitemia levels ranging from only 16 to 20,200 parasites/μl for P. falciparum disease and from 114 to 11,000 parasites/μl for P. vivax malaria. We demonstrate that the use of nested PCR allows the detection of 0.005 parasites/μl without the use of radioactive material. The use of a 1-ml sample volume and the organic DNA extraction method should be suitable in blood banks and for the evaluation of patients during and after drug treatment.

Antimalarial drug susceptibility testing of Plasmodium falciparum in Brazil using a radioisotope method

From March 1996 to August 1997, a study was carried out in a malaria endemic area of the Brazilian Amazon region. In vivo sensitivity evaluation to antimalarial drugs was performed in 129 patients. Blood samples (0.5 ml) were drawn from each patient and cryopreserved to proceed to in vitro studies. In vitro sensitivity evaluation performed using a radioisotope method was carried out with the cryopreserved samples from September to December 1997. Thirty-one samples were tested for chloroquine, mefloquine, halofantrine, quinine, arteether and atovaquone. Resistance was evidenced in 96.6% (29/30) of the samples tested for chloroquine, 3. 3% (1/30) for quinine, none (0/30) for mefloquine and none for halofantrine (0/30). Overall low sensitivity was evidenced in 10% of the samples tested for quinine, 22.5% tested for halofantrine and in 20% tested for mefloquine. Means of IC 50 values were 132.2 (SD: 46. 5) ng/ml for chloroquine, 130.6 (SD: 49.6) ng/ml for quinine, 3.4 (SD: 1.3) ng/ml for mefloquine, 0.7 (SD: 0.3) ng/ml for halofantrine, 1 (SD: 0.6) ng/ml for arteether and 0.4 (SD: 0.2) ng/ml for atovaquone. Means of chloroquine IC 50 of the tested samples were comparable to that of the chloroquine-resistant strain W2 (137.57 ng/ml) and nearly nine times higher than that of the chloroquine-sensitive strain D6 (15.09 ng/ml). Means of quinine IC 50 of the tested samples were 1.7 times higher than that of the low sensitivity strain W2 (74.84 ng/ml) and nearly five times higher than that of the quinine-sensitive strain D6 (27.53 ng/ml). These results disclose in vitro high resistance levels to chloroquine, low sensitivity to quinine and evidence of decreasing sensitivity to mefloquine and halofantrine in the area under evaluation.

Standardization of a very specific and sensitive single PCR for detection of Plasmodium vivax in low parasitized individuals and its usefulness for screening blood donors

Here, we describe the standardization of a very sensitive and specific single Plasmodium vivax polymerase chain reaction (PCR) and its usefulness for diagnosis and screening procedures when a Plasmodium falciparum PCR was also utilized. The P. vivax PCR sensitivity threshold was 0.019 parasites per microliter, and a PCR fragment was only detected when P. vivax DNA was present. Among the 11 febrile patients with negative parasitological examination that attended the malaria service of Fundação de Medicina Tropical do Amazonas, we diagnosed one P. vivax malaria by PCR. Among the 286 individuals considered suitable for blood donation, we also detected by PCR an individual with P. vivax malaria, and conversely, we did not detect any malaria infection in blood donor candidates considered unsuitable due to its past malaria history. We conclude that PCR is the method of choice for low-parasitized individuals and could therefore represent a complementary tool to safely rescue blood donor candidates considered unsuitable on the basis of malaria history.

Population Genetics of GYPB and Association Study between GYPB*S/s Polymorphism and Susceptibility to P. falciparum Infection in the Brazilian Amazon

Background Merozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil. Methods Two groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection. Results GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity. Conclusion Epidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is associated with the GPB S+ variant in this population.

Induction of cell death on Plasmodium falciparum asexual blood stages by Solanum nudum steroids

Solanum nudum Dunal (Solanaceae) is a plant used in traditional medicine in Colombian Pacific Coast, from which five steroids denominated SNs have been isolated. The SNs compounds have antiplasmodial activity against asexual blood stages of Plasmodium falciparum strain 7G8 with an IC(50) between 20-87microM. However, their mode of action is unknown. Steroids regulate important cellular functions including cell growth, differentiation and death. Thus, the aim of this work was to determine the effects of S. nudum compounds on P. falciparum asexual blood stages and their association with cell death. We found that trophozoite and schizont stages were the most sensitive to SNs. By Giemsa-stained smears, induction of crisis forms was observed. Transmission electron microscopy of treated parasites showed morphological abnormalities such as a cytoplasm rich in vesicles and myelinic figures. The Mitochondria presented no morphological alterations and the nuclei showed no abnormal chromatin condensation. By the use of S. nudum compounds, cell death in P. falciparum was evident by a decrease in mitochondrial membrane potential, DNA fragmentation and cytoplasmic acidification. The asexual blood stages of P. falciparum showed some apoptotic-like and autophagic-like cell death characteristics induced by SNs treatment.