MOESM6 of Genomic variation in two gametocyte non-producing Plasmodium falciparum clonal lines
Susana CampinoErnest Diez BenaventeSamuel AssefaEloise ThompsonLaura DroughtCatherine TaylorZaria GorvettCéline CarretChristian FlueckAl IvensDominic KwiatkowskiPietro AlanoDavid BakerTaane G. Clark
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Conference Abstract| July 01 1993 Clonal Antigenic Variation in P. Falciparum DJ Roberts; DJ Roberts 1Molecular Parasitology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9 DU, Birmingham B15 2TT Search for other works by this author on: This Site PubMed Google Scholar AG Craig; AG Craig 1Molecular Parasitology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9 DU, Birmingham B15 2TT Search for other works by this author on: This Site PubMed Google Scholar AR Berendt; AR Berendt 1Molecular Parasitology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9 DU, Birmingham B15 2TT Search for other works by this author on: This Site PubMed Google Scholar G Nash; G Nash *University of Birmingham, Birmingham B15 2TT Search for other works by this author on: This Site PubMed Google Scholar R Pinches; R Pinches 1Molecular Parasitology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9 DU, Birmingham B15 2TT Search for other works by this author on: This Site PubMed Google Scholar K Marsh; K Marsh 1Molecular Parasitology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9 DU, Birmingham B15 2TT Search for other works by this author on: This Site PubMed Google Scholar C Newbold C Newbold 1Molecular Parasitology Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9 DU, Birmingham B15 2TT Search for other works by this author on: This Site PubMed Google Scholar Clin Sci (Lond) (1993) 85 (s29): 30P–31P. https://doi.org/10.1042/cs085030Pc Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Twitter LinkedIn Cite Icon Cite Get Permissions Citation DJ Roberts, AG Craig, AR Berendt, G Nash, R Pinches, K Marsh, C Newbold; Clonal Antigenic Variation in P. Falciparum. Clin Sci (Lond) 1 July 1993; 85 (s29): 30P–31P. doi: https://doi.org/10.1042/cs085030Pc Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsClinical Science Search Advanced Search This content is only available as a PDF. © 1993 The Biochemical Society and the Medical Research Society1993 Article PDF first page preview Close Modal You do not currently have access to this content.
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Determination of the number of parasite clones present in a malaria infection is usually based on Polymerase Chain Reaction (PCR) amplification of Plasmodium polymorphic genomic sequences from peripheral blood. This method however does not provide information on the developmental stages of the parasites detected, or on the potential trasmissibility of the detected genotypes to the Anopheles vector. Reverse Transcriptase-PCR assays on P. falciparum mRNAs produced specifically in sexual stages have been developed in the past few years in order to detect and genotype circulating gametocytes, the parasite transmission stages, and are discussed in this review. Assays based on P. falciparum gamete-specific gene pfs25 and gametocyte-specific polymorphic gene pfg377 can detect presence of subpatent gametocytes in infected blood, can identify the pfg377 allele(s) specifically carried by the sexual stages, and detect coexistance of gametocytes of different genotypes. These assay have been used for the first time in field studies in a region of Sudan where malaria is seasonal, and they characterised parasite clonality and pattern of gametocyte production in the subpatent parasitaemias observed in the long malaria-free season. The method of specifically detecting and genotyping gametocytes in natural infections is proving to be a useful tool in investigating parasite transmission dynamics in field studies. This approach can be further improved by developing a multilocus RT-PCR assay which includes additional polymorphic gametocyte-specific transcripts. Candidate genes can be identified from the available data on the P. falciparum genome sequence and from recent analyses of parasite stage-specific transcriptomes.
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Background. Differentiation between gametocyte-producing Plasmodium falciparum clones depends on both high levels of stage-specific transcripts and high genetic diversity of the selected genotyping marker obtained by a high-resolution typing method. By analyzing consecutive samples of one host, the contribution of each infecting clone to transmission and the dynamics of gametocyte production in multiclone infections can be studied. Methods. We have evaluated capillary electrophoresis based differentiation of 6 length-polymorphic gametocyte genes. RNA and DNA of 25 µL whole blood from 46 individuals from Burkina Faso were simultaneously genotyped. Results. Highest discrimination power was achieved by pfs230 with 18 alleles, followed by pfg377 with 15 alleles. When assays were performed in parallel on RNA and DNA, 85.7% of all pfs230 samples and 59.5% of all pfg377 samples contained at least one matching genotype in DNA and RNA. Conclusions. The imperfect detection in both, DNA and RNA, was identified as major limitation for investigating transmission dynamics, owing primarily to the volume of blood processed and the incomplete representation of all clones in the sample tested. Abundant low-density gametocyte carriers impede clone detectability, which may be improved by analyzing larger volumes and detecting initially sequestered gametocyte clones in follow-up samples.
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Additional file 1: Table S1. SNPs identified in the three lines 3D7A, A4 and F12.
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Additional file 4: Table S4. Structural variants identified in the three lines 3D7A, A4 and F12.
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Abstract Plasmodium gametocytes are the sexual forms of the malaria parasite essential for transmission to mosquitoes. To better understand how gametocytes differ from asexual blood-stage parasites, we performed a systematic analysis of available ‘omics data for P. falciparum and other Plasmodium species. 18 transcriptomic and proteomic data sets were evaluated for the presence of curated “gold standards” of 41 gametocyte-specific versus 46 non-gametocyte genes and integrated using Bayesian probabilities, resulting in gametocyte-specificity scores for all P. falciparum genes. To illustrate the utility of the gametocyte score, we explored newly predicted gametocyte-specific genes as potential biomarkers of gametocyte carriage and exposure. We analyzed the humoral immune response in field samples against 30 novel gametocyte-specific antigens and found five antigens to be differentially recognized by gametocyte carriers as compared to malaria-infected individuals without detectable gametocytes. We also validated the gametocyte-specificity of 15 identified gametocyte transcripts on culture material and samples from naturally infected individuals, resulting in eight transcripts that were >1000-fold higher expressed in gametocytes compared to asexual parasites and whose transcript abundance allowed gametocyte detection in naturally infected individuals. Our integrated genome-wide gametocyte-specificity scores provide a comprehensive resource to identify targets and monitor P. falciparum gametocytemia.
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Gametocyte production by cloned lines of Plasmodium falciparum and their parental isolates has been studied in culture over periods of several months. Many isolates differed significantly from each other in their capacity for gametocyte production. Clones derived from an individual isolate were also widely different in capacity for gametocyte production. Consistent differences in gametocyte production were observed between clones which had always been grown concurrently and thus had identical culture histories. Levels of gametocytogenesis characteristic of individual clones, although subject to transient fluctuations under environmental influence, were stable over several months.
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