Short report: rare Plasmodium falciparum merozoite surface protein 1 19-kda (msp-1(19)) haplotypes identified in Mali using high-throughput genotyping methods

Genetic diversity in malaria vaccine antigens may compromise malaria vaccine efficacy, so it is important to understand this diversity and the processes that generate it. By applying new high-throughput genotyping methods to a large sample of infections from Mali (N = 1369), seven new 19-kDa merozoite surface protein 1 (MSP-1(19)) haplotypes were identified. Herein we report the sequences of these new haplotypes and discuss their possible origins. Although they are present in < 1% of the samples examined, the existence of these rare haplotypes reveals a greater degree of diversity at this locus than previously reported and highlights the potential for Plasmodium to evolve under selective pressure from the immune system and from such interventions as vaccines and drugs.     

Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria.

Molecular assays for monitoring sulfadoxine-pyrimethamine-resistant Plasmodium falciparum have not been implemented because of the genetic and statistical complexity of the parasite mutations that confer resistance and their relation to treatment outcomes. This study analyzed pretreatment dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes and treatment outcomes in a double-blind, placebo-controlled trial of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment for uncomplicated P. falciparum malaria. Multiple logistic regression was used to identify mutations that were predictive of treatment failure and to identify interactions and confounding factors. Infections caused by parasites with 3 DHFR mutations and 2 DHPS mutations (the "quintuple mutant") were associated with sulfadoxine-pyrimethamine treatment failure but not with chlorproguanil-dapsone treatment failure. The presence of a single DHFR mutation (Arg-59) with a single DHPS mutation (Glu-540) accurately predicted the presence of the quintuple mutant. If this model is validated in other populations, it will finally be possible to use molecular markers for surveillance of antifolate-resistant P. falciparum malaria in Africa.     

Blood folate concentrations and in vivo sulfadoxine-pyrimethamine failure in Malawian children with uncomplicated Plasmodium falciparum malaria

Folate antagonizes the antimalarial action of sulfadoxine-pyrimethamine (SP) in vitro, but its role in vivo is not well understood. We measured blood folate concentrations and SP therapeutic outcomes in Malawian children. Children with late treatment failure and those with adequate clinical and parasitologic responses had similar demographic characteristics, prevalence of parasite mutations conferring resistance to SP, and blood concentrations of anti-malarial drugs following treatment. However, a higher folate concentration was associated with late treatment failure. Patients from a low malaria transmission site had higher blood folate concentrations than those in a higher transmission site (mean +/- SEM = 39 +/- 9.3 ng/mL versus 29 +/- 10 ng/mL; P < 0.0001), and there was a higher rate of late treatment failure in the low transmission area (54.4% versus 40.2%; P = 0.010). This study also provides the first evidence of the independent role of physiologic folate concentrations in in vivo SP therapeutic efficacy, and the critical role of pyrimethamine concentrations in the therapeutic efficacy of SP when one controls physiologic folate levels and the frequency of critical dihydrofolate reductase/dihydropteroate synthase mutations.     

Immunohistochemical characterization of a 183 KD myeloid-specific-DNA- binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1

A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.     

Guidelines for international collaborative research

OBJECTIVE: As the global village becomes a reality, there is an increasing need to conduct international collaborative studies in family practice. A workshop at the WONCA meeting in Hong Kong used international attendees to produce a set of guidelines for international research. METHODS: At the workshop four completed international projects, each using a different strategy, were presented so that common themes might become apparent. The themes were then discussed and guidelines emerged from the process. RESULTS: Seven guidelines emerged for consideration before embarking on an international collaborative research project in family medicine. The guidelines deal with the characteristics of the research question and the importance of communication. The need for simple, brief methods of data collection, funding and pilot testing were identified. CONCLUSION: The question must be relevant to all participants to maintain interest and measurement tools must be validated to understand the impact of cultural differences in understanding.      

P. falciparum dihydrofolate reductase and dihydropteroate synthase mutations: epidemiology and role in clinical resistance to antifolates

Plasmodium falciparum resistance to the antifolates has arisen rapidly in Asia and South America, and threatens the usefulness of these drugs in Africa. In vitro resistance to the antifolates is determined by mutations in parasite dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS). The role of DHFR and DHPS mutations in therapeutic failure of antifolate antimalarials is less clear. This review summarizes molecular epidemiological surveys, studies of in vivo selection of mutant alleles by drug treatment, and prospective studies of the ability of mutation-specific assays to predict clinical outcomes, and discusses the potential use of these assays for surveillance of resistance.