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D F Wirth

Publications and source records attributed to D F Wirth.

At least 19 recordsLinked to original sources
PubMed2006

Defining the origin of Plasmodium falciparum resistant dhfr isolates in Senegal.

We previously reported a high baseline prevalence of mutations in the dhfr and dhps genes of Plasmodium falciparum throughout Senegal. The highest prevalence of the triple dhfr pyrimethamine associated mutations were found in isolates obtained in the western part of the country near the capital city of Dakar. In this study, we sought out to determine the relatedness of dhfr wild type and mutated strains by analyzing three microsatellite regions upstream of the dhfr locus. Twenty-six of the 31 wild type strains had a unique microsatellite pattern. In contrast, of the 17 isolates containing the triple mutation in dhfr, 11 had an identical microsatellite pattern. Diverse geographical isolates in Senegal containing the triple dhfr mutation have arisen from a limited number of ancestral strains. In addition, we demonstrate that these isolates have shared ancestry with the previously reported triple mutation haplotype found in Tanzania, South Africa, and southeast Asia. This common ancestry may have implications for the malaria control strategy for reducing the spread of sulfadoxine-pyrimethamine resistance in Senegal and elsewhere in Africa.

Animals↗
PubMed2006

Linkage disequilibrium between two distinct loci in chromosomes 5 and 7 of Plasmodium falciparum and in vivo chloroquine resistance in Southwest Nigeria.

Chloroquine (CQ) resistance in Plasmodium falciparum is associated with polymorphisms in loci on pfcrt and pfmdr1 genes. In this study, we determined the association and linkage disequilibrium between in vivo CQ resistance and P. falciparum polymorphisms in pfcrt gene at codon 76 and pfmdr1 gene at codon 86 in isolates obtained from 111 children with acute uncomplicated falciparum malaria in Nigeria. Patients were treated with standard dosage of CQ and followed up for 28 days. Filter paper samples were collected at enrollment and during follow-up for parasites genotypes and identification of pfcrt and pfmdr1 mutations. Association and linkage disequilibrium between mutant pfcrtT76 and pfmdr1Y86 alleles in pretreatment isolates of P. falciparum was determined. Fifty-five out of the 111 patients (49.5%) failed treatment. Single mutant pfcrtT76 or pfmdr1Y86 alleles were found in 55 out of 111 P. falciparum isolates screened at enrollment. Of these 55 isolates, the mutant pfcrtT76 and pfmdr1Y86 alleles were found in 84%. Both mutant pfcrtT76 (p=0.0196) and pfmdr1Y86 (p=0.000042) alleles were associated with in vivo CQ resistance. In addition, the mutant pfcrtT76 (p=0.047) and pfmdr1Y86 (p=0.006) alleles were significantly selected by CQ in patients who failed treatment. Association analysis between paired single alleles at pfcrt and pfmdr1 loci showed a significant association (p=0.0349 and chi(2)=4.45) between the pfcrt T76 allele on chromosome 7 and the pfmdr1Y86 allele on chromosome 5 and that these two mutant alleles were in linkage disequilibrium (p=0.000, D'=0.64, and r(2)=0.28). Considering the high level of CQ resistance and drug use in the study area, the observed linkage disequilibrium between the mutant pfcrtT76 and pfmdr1Y86 alleles is maintained epistatically through directional CQ selective pressure.

Animals↗
PubMed2006

Association between mutations in Plasmodium falciparum chloroquine resistance transporter and P. falciparum multidrug resistance 1 genes and in vivo amodiaquine resistance in P. falciparum malaria-infected children in Nigeria.

This study investigated the association between Plasmodium falciparum chloroquine resistance transporter (pfcrt) T76 and P. falciparum multidrug resistance gene 1 (pfmdr1) Y86 alleles and in vivo amodiaquine (AQ) resistance, as well as the clearance of parasites harboring these two alleles in children treated with AQ in southwest Nigeria. One hundred one children with acute uncomplicated P. falciparum malaria infections were treated with the standard dosage of AQ and followed-up for 28 days. Blood samples were collected on filter paper samples at enrollment and during follow-up for identification of parasite genotypes and pfcrt and pfmdr1 mutations using polymerase chain reaction and restriction fragment length polymorphism approaches. Parasitologic assessment of response to treatment showed that 87% and 13% (RI) of patients were cured and failed treatment, respectively. Although infections in patients were polyclonal (as determined by merozoite surface protein 2 genotyping), the presence of both mutants pfcrtT76 and pfmdr1Y86 alleles in parasites is associated with in vivo AQ resistance (odds ratio = 7.58, 95% confidence interval = 1.58-36.25, P = 0.006) and is selected by the drug in children who failed AQ treatment. Treatment failure with the combination of mutant pfcrtT76 and pfmdr1Y86 alleles as well as the ability of patients to clear these resistant parasites is dependent on age, suggesting a critical role of host immunity in clearing AQ-resistant P. falciparum. The combination of mutant pfcrtT76 and pfmdr1Y86 alleles may be useful markers for monitoring the development and spread of AQ resistance, when combining this drug with other antimalarials for treatment of malaria in Africa.

Age Factors↗
PubMed2005

Polymorphisms in Plasmodium falciparum dhfr and dhps genes and age related in vivo sulfadoxine-pyrimethamine resistance in malaria-infected patients from Nigeria.

Mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes have been used as means to predict treatment failure to sulfadoxine-pyrimethamine (SP) and for monitoring/surveillance of resistance to the drug in many areas where malaria is endemic. However, patients responses to treatment are significantly dependent on factors like host immunity profile of treated patients. In order to investigate the relationship between molecular markers of SP resistance, host immunity and clinical outcome, the association between pre-treatment dhfr and dhps genotypes, age and treatment outcomes was evaluated in 109 children treated with SP for acute uncomplicated malaria in Ibadan, Nigeria. Seventy-three percent of the children were cured with the drug, while 27% failed treatment after 28 days of follow-up. All children infected with parasites harboring less than two dhfr/dhps mutations were cured with SP. The dhfr triple (Asn-108/Ile-51/Arg-59) mutants or the dhps double mutants (Gly-437/Glu-540) were independently associated with SP treatment failure in children aged less than 5 years, but not in older children. The dhfr and dhps quintuple mutant (dhfr triple mutant+dhps double mutant) was the genotype most strongly associated with SP treatment failure (OR=24.72, 95%CI=8.24-74.15) in both younger and older children.

Animals↗
PubMed2005

Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase genes in Senegal.

Senegal recently (2004) switched to sulfadoxine-pyrimethamine (SP) with amodiaquine as first line therapy for malaria in response to increasing chloroquine resistance. In anticipation of emerging resistance to SP as a result of this change in drug pressure, we set out to define the baseline prevalence of SP-associated mutations in the dhfr and dhps genes in Plasmodium falciparum using geographically diverse and longitudinally collected samples. A total of 153 blood samples were analysed from patients (5 years or older) with mild malaria after informed consent was obtained. Longitudinal samples were collected between 2000 and 2003 in Pikine, a suburb of Dakar. Geographically diverse site sampling was carried out in 2003. The mutation prevalence in DHFR codons 51, 59 and 108 is 65%, 61% and 78% in Pikine, 2003. The overall prevalence of the triple mutation that is associated with high-level pyrimethamine resistance is 61%. The mutation prevalence rate in DHPS codons 436 and 437 is 21% and 40%, respectively. There is significant geographic variation in genotypic resistance, as samples from Pikine in 2003 had higher mutation prevalence in the pfdhfr and pfdhps genes compared to samples from Tambacounda (P < 0.015). In summary, this study demonstrates a high background prevalence of SP resistance mutations already present in P. falciparum in Senegal.

Animals↗
PubMed2004

Molecular analysis of Plasmodium falciparum recrudescent malaria infections in children treated with chloroquine in Nigeria.

Parasite genotyping by a polymerase chain reaction was used to distinguish recrudescent from newly acquired Plasmodium falciparum infections in 50 of 160 Nigerian children taking part in a chloroquine efficacy study in Ibadan, Nigeria. A finger prick blood sample was taken from each child before and after treatment to identify recrudescent parasites. By investigating allelic variation in three polymorphic antigen loci, merozoite surface protein-1 (MSP-1), MSP-2, and glutamate-rich protein (GLURP), we determined parasite diversity in the population and in the infected host. DNA from pretreatment and post-treatment samples from 47 of the 50 patients who failed therapy was successfully amplified by the PCR. The MSP-1, MSP-2, and GLURP genotypes in all samples showed extensive diversity, indicating polyclonal infections. The average number of clones per infection in pre-treatment sample was 2.5 with MSP-1, 4.9 with MSP-2, and 2 with GLURP. The extent of multiplicity decreased significantly (P = 0.016) in posttreatment samples. Multiplicity of infection and initial parasite density were not age dependent. Comparison of the variant alleles in pretreatment and post-treatment samples of each patient indicates that 26 of the 47 children had genuinely recrudescent disease. Conversely, post-treatment samples from five children showed completely new genotypes, indicating either a previously sequestered population of parasites or a newly acquired infection. Overall, this study has shown the diversity and complexity of P. falciparum population in Ibadan, Nigeria. The study has also shown the dynamics of P. falciparum infections in this population before and after chloroquine treatment in an area of high malaria transmission.

Animals↗
PubMed2003

Prevalence of Plasmodium falciparum pfcrt polymorphisms and in vitro chloroquine sensitivity in Senegal.

Mutations in pfcrt K76T are associated with chloroquine resistance in Plasmodium falciparum. Previous studies of K76T mutations in Senegal reported the association of T76 with in vitro-resistant isolates, but this mutation was also prevalent in chloroquine-sensitive isolates. This suggests involvement of additional genetic loci in modulating chloroquine resistance. Additional pfcrt polymorphisms at codons A220S, Q271E, N326S and R371I have been found in chloroquine-resistant isolates. We wanted to test if sequential acquisition of mutations at these codons leads to in vitro chloroquine resistance. Stepwise accumulation of mutations was not detected, rather there was almost complete linkage between the pfcrt K76T mutation and polymorphisms in these codons. Therefore these additional polymorphisms do not enhance the correlation between pfcrt T76 and chloroquine resistance in Senegal. These data suggest that in vitro chloroquine resistance requires the genetic background of the pfcrt K76T mutation and additional mutations in genetic loci outside the pfcrt gene.

Adolescent↗
PubMed2003

Point mutations in the pfcrt and pfmdr-1 genes of Plasmodium falciparum and clinical response to chloroquine, among malaria patients from Nigeria.

Chloroquine (CQ) resistance in Plasmodium falciparum has been associated with specific point mutations in the pfcrt and pfmdr-1 genes. In the present study, 30 children aged 1-12 years, who were all suffering from acute, uncomplicated, P. falciparum malaria in Ibadan, Nigeria, were evaluated to assess the association between these mutations and clinical outcome following treatment with CQ. The parasites, in blood samples collected pre-treatment and, in those who failed treatment, on the day symptoms re-occurred post-treatment, were genotyped using the polymorphic MSP1, MSP2 and GLURP loci and PCR-RFLP. The results showed that, pre-treatment, all 30 patients had polyclonal infections, the mean numbers of P. falciparum clones detected per infection being 2.6 with MSP1, 4.2 with MSP2 and 2.8 with GLURP. The T76 allele of pfcrt and the Y86 allele of pfmdr-1 were found in 53% and 40%, respectively, of the pre-treatment samples from the 15 patients who failed CQ treatment, but the Y1246 mutation in pfmdr-1 was never detected. Although the parasites from the two patients with high-grade (RIII) resistance to CQ had both of these point mutations, the presence of the T76 allele of pfcrt or the Y86 allele of pfmdr-1 (considered individually) could not be used to predict treatment outcome. However, a high frequency of clonal multiplicity may confound attempts to associate the point mutations in pfcrt or pfmdr-1 with clinical response to CQ. It remains unclear whether the present results represent the characteristics of the predominant parasite populations in the study area. Further studies are needed before the strength of the association between the point mutations identified as markers of drug resistance and clinical outcome can be accurately evaluated, in this and other regions of intense transmission.

ATP-Binding Cassette Transporters↗
PubMed2001

Recent origin of Plasmodium falciparum from a single progenitor.

Genetic variability of Plasmodium falciparum underlies its transmission success and thwarts efforts to control disease caused by this parasite. Genetic variation in antigenic, drug resistance, and pathogenesis determinants is abundant, consistent with an ancient origin of P. falciparum, whereas DNA variation at silent (synonymous) sites in coding sequences appears virtually absent, consistent with a recent origin of the parasite. To resolve this paradox, we analyzed introns and demonstrated that these are deficient in single-nucleotide polymorphisms, as are synonymous sites in coding regions. These data establish the recent origin of P. falciparum and further provide an explanation for the abundant diversity observed in antigen and other selected genes.

Africa↗
PubMed2001

Serial analysis of gene expression (SAGE) in Plasmodium falciparum: application of the technique to A-T rich genomes.

The advent of high-throughput methods for the analysis of global gene expression, together with the Malaria Genome Project open up new opportunities for furthering our understanding of the fundamental biology and virulence of the malaria parasite. Serial analysis of gene expression (SAGE) is particularly well suited for malarial systems, as the genomes of Plasmodium species remain to be fully annotated. By simultaneously and quantitatively analyzing mRNA transcript profiles from a given cell population, SAGE allows for the discovery of new genes. In this study, one reports the successful application of SAGE in Plasmodium falciparum, 3D7 strain parasites, from which a preliminary library of 6880 tags corresponding to 4146 different genes was generated. It was demonstrated that P. falciparum is amenable to this technique, despite the remarkably high A-T content of its genome. SAGE tags as short as 10 nucleotides were sufficient to uniquely identify parasite transcripts from both nuclear and mitochondrial genomes. Moreover, the skewed A-T content of parasite sequence did not preclude the use of enzymes that are crucial for generating representative SAGE libraries. Finally, a few modifications to DNA extraction and cloning steps of the SAGE protocol proved useful for circumventing specific problems presented by A-T rich genomes.

Animals↗
PubMed2001

Serial analysis of gene expression in Plasmodium falciparum reveals the global expression profile of erythrocytic stages and the presence of anti-sense transcripts in the malarial parasite.

Serial analysis of gene expression (SAGE) was applied to the malarial parasite Plasmodium falciparum to characterize the comprehensive transcriptional profile of erythrocytic stages. A SAGE library of approximately 8335 tags representing 4866 different genes was generated from 3D7 strain parasites. Basic local alignment search tool analysis of high abundance SAGE tags revealed that a majority (88%) corresponded to 3D7 sequence, and despite the low complexity of the genome, 70% of these highly abundant tags matched unique loci. Characterization of these suggested the major metabolic pathways that are used by the organism under normal culture conditions. Furthermore several tags expressed at high abundance (30% of tags matching to unique loci of the 3D7 genome) were derived from previously uncharacterized open reading frames, demonstrating the use of SAGE in genome annotation. The open platform "profiling" nature of SAGE also lead to the important discovery of a novel transcriptional phenomenon in the malarial pathogen: a significant number of highly abundant tags that were derived from annotated genes (17%) corresponded to antisense transcripts. These SAGE data were validated by two independent means, strand specific reverse transcription-polymerase chain reaction and Northern analysis, where antisense messages were detected in both asexual and sexual stages. This finding has implications for transcriptional regulation of Plasmodium gene expression.

Animals↗
PubMed2000

3' UTR elements enhance expression of Pgs28, an ookinete protein of Plasmodium gallinaceum.

In Plasmodium parasites the fusion of gametes to form a fertilized zygote and morphogenesis into the motile ookinete are critical developmental stages in the parasite's complex life cycle. In analogous developmental stages of metazoan organisms 3' gene flanking regions are critical in the regulation of gene expression. To determine whether these mechanisms are conserved in the protozoan parasite we studied the 3' gene flanking elements necessary for the expression of Pgs28, the major surface protein of mature zygotes and ookinetes of the chicken malaria Plasmodium gallinaceum. The DNA sequence of the pgs28 3' gene flanking region contains 7 eukaryotic polyadenylation consensus signals (AATAAA/ATTAAA). An unusual 82% T-rich region is located 55 nucleotides upstream of the fifth polyadenylation signal (ATTAAA). The pgs28 mRNA terminates approximately 20 nucleotides from the polyadenylation signal in a poly (A) tail. To determine whether the T-rich region and polyadenylation signals were necessary for Pgs28 protein expression, sexual stage parasites were transfected with plasmids containing deletions of these elements utilizing firefly luciferase (LUC) and beta-glucuronidase (GUS) as markers of transient gene transfection. The parasites were allowed to develop in vitro to the ookinete stage and assayed for enzymatic activity. Cells transfected with plasmids containing deletions of the T-rich region or fifth eukaryotic polyadenylation consensus signal expressed 89 and 92%, less enzymatic activity respectively than those transfected with the full length pgs28 3' gene flanking region. The U-rich element and fifth eukaryotic polyadenylation consensus sequence within the pgs28 3' UTR are therefore necessary for Pgs28 protein expression.

3' Untranslated Regions↗
PubMed2000

The signal sequence and C-terminal hydrophobic domain are required for localization of the sexual stage antigen pgs28 to the surface of P. gallinaceum ookinetes.

The Pgs28 protein is a major surface antigen of the sexual stages of Plasmodium gallinaceum the zygotes and the ookinetes. The protein contains conserved motifs, namely an N-terminal signal sequence, four epidermal growth factor-like repeats and a C-terminal hydrophobic domain that serves as a signal for glycosylphosphatidylinositol (GPI)--anchor modification. In this study, we define the protein motifs required for the surface localization of Pgs28 in ookinetes. using transient transfection combined with immunofluorescence and confocal microscopy. Pgs28 fused to the green fluorescent protein (Pgs28-GFP) is expressed in zygotes, intermediate retort forms and ookinetes. Mutational analyses of Pgs28 coding regions reveal that deletions of the signal sequence and the C-terminal domain result in intracellular retention of the fusion protein. Therefore, the signal sequence and C-terminal domain are required for cell surface localization. Additionally, the Pgs28-GFP fusion proteins are shed from the surface of live ookinetes, suggesting that Pgs28 may be involved in interactions with the cells of the mosquito midgut or during motility.

Animals↗
PubMed2000

Gender distribution in asymptomatic and invasive amebiasis.

OBJECTIVE: The majority of individuals infected by the protozoan parasite Entamoeba histolytica experience subclinical infections. However, a small proportion of parasitized individuals develop severe invasive disease such as amebic dysentery or amebic liver abscess. Invasive amebiasis affects predominantly men; the usual explanation for this has been that men have a higher rate of asymptomatic infections and therefore experience a higher rate of invasive disease. To date, there is no convincing evidence of an increased rate of asymptomatic infection of men as compared with women. The purpose of this study was to evaluate the evidence supporting the hypothesis that men have higher rates of asymptomatic infection and thus an increased frequency of invasive amebiasis. METHODS: We reviewed published reports of invasive amebiasis and population-based parasitological studies from 1929-1997 to compare the gender ratio of asymptomatic and symptomatic E. histolytica infection. Infections with E. histolytica were differentiated from the nonpathogenic E. dispar whenever possible. RESULTS: The reports of invasive amebiasis (dysentery, liver abscess, colonic perforation, peritonitis, appendicitis, and ameboma) showed a higher proportion of men than women (ratio, male:female = 3.2:1, p < 0.05). This contrasts with the epidemiological surveys, where the rate of asymptomatic infection with E. histolytica was the same (1:1) for both genders (p > 0.05). CONCLUSIONS: Asymptomatic E. histolytica infection is equally distributed between the genders. The high proportion of men with invasive amebiasis may be due to a male-related susceptibility to invasive disease.

Adult↗
PubMed2000

Technical assessment of the affymetrix yeast expression GeneChip YE6100 platform in a heterologous model of genes that confer resistance to antimalarial drugs in yeast.

The advent of high-density gene array technology has revolutionized approaches to drug design, development, and characterization. At the laboratory level, the efficient, consistent, and dependable exploitation of this complex technology requires the stringent standardization of protocols and data analysis platforms. The Affymetrix YE6100 expression GeneChip platform was evaluated for its performance in the analysis of both global (6,000 yeast genes) and targeted (three pleiotropic multidrug resistance genes of the ATP binding cassette transporter family) gene expression in a heterologous yeast model system in the presence and absence of the antimalarial drug chloroquine. Critical to the generation of consistent data from this platform are issues involving the preparation of the specimen, use of appropriate controls, accurate assessment of experiment variance, strict adherence to optimized enzymatic and hybridization protocols, and use of sophisticated bioinformatics tools for data analysis.

ATP-Binding Cassette Transporters↗
PubMed1999

Genetic characterization of an epidemic of Plasmodium falciparum malaria among Yanomami Amerindians.

Malaria parasites are genetically diverse at all levels of endemicity. In contrast, the merozoite surface protein (MSP) alleles in samples from 2 isolated populations of Yanomami Amerindians during an epidemic of Plasmodium falciparum were identical. The nonvariable restriction fragment length polymorphism patterns further suggested that the sequential outbreak comprised only a single P. falciparum genotype. By examination of serial samples from single human infections, the MSP characteristics were found to remain constant throughout the course of infection. An apparent clonal population structure of parasites seemed to cause outbreaks in small isolated villages. The use of standard molecular epidemiologic methods to measure genetic diversity in malaria revealed the occurrence of a genetically monomorphic population of P. falciparum within a human community.

Animals↗