Dr Olivo Miotto
|Research Area:||Global Health|
|Technology Exchange:||Bioinformatics, Computational biology, SNP typing and Statistical genetics|
|Scientific Themes:||Genetics & Genomics and Tropical Medicine & Global Health|
|Keywords:||genomics, informatics, malaria, epidemiology, infectious disease and software|
Because of malaria, millions of African children die before their first day at school. For decades, scientists and health authorities have raised hopes of eliminating this burden on the developing world, only to have these hopes dashed, time after time, by the astonishing adaptability of Plasmodium parasites, which seem capable of becoming tolerant to most drugs we deploy. Today, genomic technologies provide a powerful new weapon against these parasites. At the Centre for Genomics and Global Health, we have developed methods for determining the genetic code of Plasmodium parasite directly from blood samples taken from infected patients. Thanks to the falling cost of sequencing, and the reliability of our approach, we are now able to sequence literally thousands of parasites from clinical cases, and map this genomic information in geographical, ecological and historical contexts. Our goal is to determine how parasite populations change as a result of human intervention and environmental change, and observe new epidemiological trends in real time, to inform and guide public health intervention. In other words, "genomic epidemiology" will monitor Plasmodium evolution, so that humanity can keep steps ahead of the parasite.
Olivo Miotto focuses on translating the massive quantities of data produced by sequencing thousands of genomes into meaningful knowledge about the epidemiology of Plasmodium falciparum. By analyzing hundreds of thousands of genomic variations in each blood sample, he studies the genetics of parasite populations in four continents, and identifies patterns of evolution associated to responses to drug pressure and other human interventions. Based in Bangkok, Olivo collaborates with many clinical research groups in malaria endemic regions, particularly in Southeast Asia, to study relationships between response to clinical therapy and genetics of the disease-causing parasites in the patient. In the early stages, the focus is on immediate problems, such as discovering mutations causing resistance to current drugs, such as artemisinin. However, Olivo's longer-term perspective is to create tools that will identify patterns in thousands of accumulated genomic sequences, leading to a deep understanding of parasite evolution and, ultimately, to interventions that will win the struggle against the disease.
When learning about Darwin at age 12, Olivo thought evolution was the coolest thing he had heard of. It took him decades to realize that it was also the best use for his skills. In between, he studied physics and worked as a software engineer, both in the commercial and academic worlds. At various times he focussed on Web applications, graphical user interfaces, speech synthesis, online learning, and database systems, before finally switching to bioinformatics and genomics.
|Professor Nicholas J White FRS||Tropical Medicine||University of Oxford||United Kingdom|
|Professor Nicholas PJ Day FMedSci FRCP||Tropical Medicine||University of Oxford||United Kingdom|
|Professor Adrianus Dondorp||Tropical Medicine||University of Oxford||United Kingdom|
|Professor Chris V Plowe||University of Maryland||United States|
|Dr Rick Fairhurst||NIAID||NIH||United States|
|Professor François H Nosten||Tropical Medicine||University of Oxford||United Kingdom|
|Dr Caterina I Fanello||Tropical Medicine||University of Oxford||United Kingdom|
|Dr Sarah Auburn||Menzies||Australia|
|Dr Julian Rayner||Wellcome Trust Sanger Institute||United Kingdom|
We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population. Hide abstract
Artemisinin resistance in Plasmodium falciparum threatens global efforts to control and eliminate malaria. Polymorphisms in the kelch domain-carrying protein K13 are associated with artemisinin resistance, but the underlying molecular mechanisms are unknown. We analyzed the in vivo transcriptomes of 1043 P. falciparum isolates from patients with acute malaria and found that artemisinin resistance is associated with increased expression of unfolded protein response (UPR) pathways involving the major PROSC and TRiC chaperone complexes. Artemisinin-resistant parasites also exhibit decelerated progression through the first part of the asexual intraerythrocytic development cycle. These findings suggest that artemisinin-resistant parasites remain in a state of decelerated development at the young ring stage, whereas their up-regulated UPR pathways mitigate protein damage caused by artemisinin. The expression profiles of UPR-related genes also associate with the geographical origin of parasite isolates, further suggesting their role in emerging artemisinin resistance in the Greater Mekong Subregion. Hide abstract
BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. METHODS: Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. RESULTS: The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. CONCLUSIONS: Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; ClinicalTrials.gov number, NCT01350856.). Hide abstract
Reduced Plasmodium falciparum sensitivity to short-course artemisinin (ART) monotherapy manifests as a long parasite clearance half-life. We recently defined three parasite founder populations with long half-lives in Pursat, western Cambodia, where reduced ART sensitivity is prevalent. Using the ring-stage survival assay, we show that these founder populations have reduced ART sensitivity in vitro at the early ring stage of parasite development and that a genetically admixed population contains subsets of parasites with normal or reduced ART sensitivity. Hide abstract
Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain ('K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread. Hide abstract
We describe an analysis of genome variation in 825 P. falciparum samples from Asia and Africa that identifies an unusual pattern of parasite population structure at the epicenter of artemisinin resistance in western Cambodia. Within this relatively small geographic area, we have discovered several distinct but apparently sympatric parasite subpopulations with extremely high levels of genetic differentiation. Of particular interest are three subpopulations, all associated with clinical resistance to artemisinin, which have skewed allele frequency spectra and high levels of haplotype homozygosity, indicative of founder effects and recent population expansion. We provide a catalog of SNPs that show high levels of differentiation in the artemisinin-resistant subpopulations, including codon variants in transporter proteins and DNA mismatch repair proteins. These data provide a population-level genetic framework for investigating the biological origins of artemisinin resistance and for defining molecular markers to assist in its elimination. Hide abstract
The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia. Hide abstract
No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC(50) values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines. Hide abstract
Malaria elimination strategies require surveillance of the parasite population for genetic changes that demand a public health response, such as new forms of drug resistance. Here we describe methods for the large-scale analysis of genetic variation in Plasmodium falciparum by deep sequencing of parasite DNA obtained from the blood of patients with malaria, either directly or after short-term culture. Analysis of 86,158 exonic single nucleotide polymorphisms that passed genotyping quality control in 227 samples from Africa, Asia and Oceania provides genome-wide estimates of allele frequency distribution, population structure and linkage disequilibrium. By comparing the genetic diversity of individual infections with that of the local parasite population, we derive a metric of within-host diversity that is related to the level of inbreeding in the population. An open-access web application has been established for the exploration of regional differences in allele frequency and of highly differentiated loci in the P.falciparum genome. © 2012 Macmillan Publishers Limited. All rights reserved. Hide abstract
Our understanding of the composition of multi-clonal malarial infections and the epidemiological factors which shape their diversity remain poorly understood. Traditionally within-host diversity has been defined in terms of the multiplicity of infection (MOI) derived by PCR-based genotyping. Massively parallel, single molecule sequencing technologies now enable individual read counts to be derived on genome-wide datasets facilitating the development of new statistical approaches to describe within-host diversity. In this class of measures the F(WS) metric characterizes within-host diversity and its relationship to population level diversity. Utilizing P. falciparum field isolates from patients in West Africa we here explore the relationship between the traditional MOI and F(WS) approaches. F(WS) statistics were derived from read count data at 86,158 SNPs in 64 samples sequenced on the Illumina GA platform. MOI estimates were derived by PCR at the msp-1 and -2 loci. Significant correlations were observed between the two measures, particularly with the msp-1 locus (P = 5.92×10(-5)). The F(WS) metric should be more robust than the PCR-based approach owing to reduced sensitivity to potential locus-specific artifacts. Furthermore the F(WS) metric captures information on a range of parameters which influence out-crossing risk including the number of clones (MOI), their relative proportions and genetic divergence. This approach should provide novel insights into the factors which correlate with, and shape within-host diversity. Hide abstract
BACKGROUND: There is widespread concern that H5N1 avian influenza A viruses will emerge as a pandemic threat, if they become capable of human-to-human (H2H) transmission. Avian strains lack this capability, which suggests that it requires important adaptive mutations. We performed a large-scale comparative analysis of proteins from avian and human strains, to produce a catalogue of mutations associated with H2H transmissibility, and to detect their presence in avian isolates. METHODOLOGY/PRINCIPAL FINDINGS: We constructed a dataset of influenza A protein sequences from 92,343 public database records. Human and avian sequence subsets were compared, using a method based on mutual information, to identify characteristic sites where human isolates present conserved mutations. The resulting catalogue comprises 68 characteristic sites in eight internal proteins. Subtype variability prevented the identification of adaptive mutations in the hemagglutinin and neuraminidase proteins. The high number of sites in the ribonucleoprotein complex suggests interdependence between mutations in multiple proteins. Characteristic sites are often clustered within known functional regions, suggesting their functional roles in cellular processes. By isolating and concatenating characteristic site residues, we defined adaptation signatures, which summarize the adaptive potential of specific isolates. Most adaptive mutations emerged within three decades after the 1918 pandemic, and have remained remarkably stable thereafter. Two lineages with stable internal protein constellations have circulated among humans without reassorting. On the contrary, H5N1 avian and swine viruses reassort frequently, causing both gains and losses of adaptive mutations. CONCLUSIONS: Human host adaptation appears to be complex and systemic, involving nearly all influenza proteins. Adaptation signatures suggest that the ability of H5N1 strains to infect humans is related to the presence of an unusually high number of adaptive mutations. However, these mutations appear unstable, suggesting low pandemic potential of H5N1 in its current form. In addition, adaptation signatures indicate that pandemic H1N1/09 strain possesses multiple human-transmissibility mutations, though not an unusually high number with respect to swine strains that infected humans in the past. Adaptation signatures provide a novel tool for identifying zoonotic strains with the potential to infect humans. Hide abstract
BACKGROUND: Genetic variation and rapid evolution are hallmarks of RNA viruses, the result of high mutation rates in RNA replication and selection of mutants that enhance viral adaptation, including the escape from host immune responses. Variability is uneven across the genome because mutations resulting in a deleterious effect on viral fitness are restricted. RNA viruses are thus marked by protein sites permissive to multiple mutations and sites critical to viral structure-function that are evolutionarily robust and highly conserved. Identification and characterization of the historical dynamics of the conserved sites have relevance to multiple applications, including potential targets for diagnosis, and prophylactic and therapeutic purposes. METHODOLOGY/PRINCIPAL FINDINGS: We describe a large-scale identification and analysis of evolutionarily highly conserved amino acid sequences of the entire dengue virus (DENV) proteome, with a focus on sequences of 9 amino acids or more, and thus immune-relevant as potential T-cell determinants. DENV protein sequence data were collected from the NCBI Entrez protein database in 2005 (9,512 sequences) and again in 2007 (12,404 sequences). Forty-four (44) sequences (pan-DENV sequences), mainly those of nonstructural proteins and representing approximately 15% of the DENV polyprotein length, were identical in 80% or more of all recorded DENV sequences. Of these 44 sequences, 34 ( approximately 77%) were present in >or=95% of sequences of each DENV type, and 27 ( approximately 61%) were conserved in other Flaviviruses. The frequencies of variants of the pan-DENV sequences were low (0 to approximately 5%), as compared to variant frequencies of approximately 60 to approximately 85% in the non pan-DENV sequence regions. We further showed that the majority of the conserved sequences were immunologically relevant: 34 contained numerous predicted human leukocyte antigen (HLA) supertype-restricted peptide sequences, and 26 contained T-cell determinants identified by studies with HLA-transgenic mice and/or reported to be immunogenic in humans. CONCLUSIONS/SIGNIFICANCE: Forty-four (44) pan-DENV sequences of at least 9 amino acids were highly conserved and identical in 80% or more of all recorded DENV sequences, and the majority were found to be immune-relevant by their correspondence to known or putative HLA-restricted T-cell determinants. The conservation of these sequences through the entire recorded DENV genetic history supports their possible value for diagnosis, prophylactic and/or therapeutic applications. The combination of bioinformatics and experimental approaches applied herein provides a framework for large-scale and systematic analysis of conserved and variable sequences of other pathogens, in particular, for rapidly mutating viruses, such as influenza A virus and HIV. Hide abstract
BACKGROUND: Influenza A viruses generate an extreme genetic diversity through point mutation and gene segment exchange, resulting in many new strains that emerge from the animal reservoirs, among which was the recent highly pathogenic H5N1 virus. This genetic diversity also endows these viruses with a dynamic adaptability to their habitats, one result being the rapid selection of genomic variants that resist the immune responses of infected hosts. With the possibility of an influenza A pandemic, a critical need is a vaccine that will recognize and protect against any influenza A pathogen. One feasible approach is a vaccine containing conserved immunogenic protein sequences that represent the genotypic diversity of all current and future avian and human influenza viruses as an alternative to current vaccines that address only the known circulating virus strains. METHODOLOGY/PRINCIPAL FINDINGS: Methodologies for large-scale analysis of the evolutionary variability of the influenza A virus proteins recorded in public databases were developed and used to elucidate the amino acid sequence diversity and conservation of 36,343 sequences of the 11 viral proteins of the recorded virus isolates of the past 30 years. Technologies were also applied to identify the conserved amino acid sequences from isolates of the past decade, and to evaluate the predicted human lymphocyte antigen (HLA) supertype-restricted class I and II T-cell epitopes of the conserved sequences. Fifty-five (55) sequences of 9 or more amino acids of the polymerases (PB2, PB1, and PA), nucleoprotein (NP), and matrix 1 (M1) proteins were completely conserved in at least 80%, many in 95 to 100%, of the avian and human influenza A virus isolates despite the marked evolutionary variability of the viruses. Almost all (50) of these conserved sequences contained putative supertype HLA class I or class II epitopes as predicted by 4 peptide-HLA binding algorithms. Additionally, data of the Immune Epitope Database (IEDB) include 29 experimentally identified HLA class I and II T-cell epitopes present in 14 of the conserved sequences. CONCLUSIONS/SIGNIFICANCE: This study of all reported influenza A virus protein sequences, avian and human, has identified 55 highly conserved sequences, most of which are predicted to have immune relevance as T-cell epitopes. This is a necessary first step in the design and analysis of a polyepitope, pan-influenza A vaccine. In addition to the application described herein, these technologies can be applied to other pathogens and to other therapeutic modalities designed to attack DNA, RNA, or protein sequences critical to pathogen function. Hide abstract
Genetic surveillance to support malaria elimination in the Greater Mekong Subregion
The malaria parasite Plasmodium falciparum causes a massive burden of disease and death throughout the developing world. The parasite is capable of rapid and extensive genetic change, undermining vaccine development with extreme antigenic diversity, and repeatedly developing resistance to antimalarial drugs. Most recently, parasites in Southeast Asia have become increasingly resistant to artemisinin, the most effective frontline drug. If it spreads to Africa, artemisinin resistance could roll ...
Artemisinin resistance surveillance using novel genetic markers
The malaria parasite Plasmodium falciparum causes a massive burden of disease and death in the developing world. Artemisinin is the main drug used to treat falciparum malaria but parasites in Southeast Asia are becoming increasingly resistant. It will be a global health disaster if artemisinin resistance spreads to Africa, where most malaria deaths occur, and an international campaign is therefore being launched to eliminate malaria in the areas where resistance is currently established. To s ...