Search

Background Brazil is a unique and understudied setting for malaria, with complex foci of transmission associated with human and environmental conditions. An understanding of the population genomic diversity of P. vivax parasites across Brazil can support malaria control strategies. Methods Through whole genome sequencing of P. vivax isolates across 7 Brazilian states, we use population genomic approaches to compare genetic diversity within country (n = 123), continent (6 countries, n = 315) and globally (26 countries, n = 885). Findings We confirm that South American isolates are distinct, have more ancestral populations than the other global regions, with differentiating mutations in genes under selective pressure linked to antimalarial drugs (pvmdr1, pvdhfr-ts) and mosquito vectors (pvcrmp3, pvP45/48, pvP47). We demonstrate Brazil as a distinct parasite population, with signals of selection including ABC transporter (PvABCI3) and PHIST exported proteins. Interpretation Brazil has a complex population structure, with evidence of P. simium infections and Amazonian parasites separating into multiple clusters. Overall, our work provides the first Brazil-wide analysis of P. vivax population structure and identifies important mutations, which can inform future research and control measures.

Chlamydia trachomatis(Ct) is the most common infectious cause of blindness and bacterial sexually transmitted infection worldwide. UsingCtwhole genome sequences obtained directly from conjunctival swabs, we studiedCtgenomic diversity and associations betweenCtgenetic polymorphisms with ocular localization and disease severity in a treatment-naïve trachoma-endemic population in Guinea Bissau, West Africa. All sequences fall within the T2 ocular clade phylogenetically. This is consistent with the presence of the characteristic deletion intrpAresulting in a truncated non-functional protein and the ocular tyrosine repeat regions present intarPassociated with ocular tissue localization. We have identified twenty-oneCtnon-synonymous single nucleotide polymorphisms (SNPs) associated with ocular localization, including SNPs withinpmpD(OR=4.07,p*=0.001) andtarP(OR=0.34,p*=0.009). Eight SNPs associated with disease severity were found inyjfH (rlmB)(OR=0.13,p*=0.037),CTA0273(OR=0.12,p*=0.027),trmD(OR=0.12,p*=0.032),CTA0744(OR=0.12,p*=0.041),glgA(OR=0.10,p*=0.026),alaS(OR=0.10,p*=0.032),pmpE(OR=0.08,p*=0.001) and the intergenic regionCTA0744-CTA0745(OR=0.13,p*=0.043). This study demonstrates the extent of genomic diversity within a naturally circulating population of ocularCt, and the first to describe novel genomic associations with disease severity. These findings direct investigation of host-pathogen interactions that may be important in ocularCtpathogenesis and disease transmission.

Background: Recent evidence suggests hospital transmission of methicillin-resistant Staphylococcus aureus (MRSA) is uncommon in UK centers that have implemented sustained infection control programmes. We investigated whether a healthcare-network analysis could shed light on transmission paths currently sustaining MRSA levels in UK hospitals. Methods: A cross-sectional observational study was performed in two NHS hospital groups and a general district hospital in South-East London. All MRSA patients identified at inpatient, outpatient and community settings between 1st November 2011 and 29th February 2012 were included. We identified genetically-defined MRSA transmission clusters in individual hospitals and across the healthcare-network, and examined genetic differentiation of ST22 MRSA isolates within and between hospitals and inpatient or outpatient and community settings, as informed by average and median pairwise single nucleotide polymorphisms (SNPs) and SNP-based proportions of nearly identical isolates (‘I’). Results: 248/610 (40.7%) MRSA patients were linked in 90 transmission-clusters, of which 27 spanned multiple hospitals. Analysis of a large 32 patient ST22-MRSA cluster showed that 26/32 patients (81·3%) had multiple contacts with one another during ward stays at any hospital. No residential, outpatient or significant community healthcare contacts were identified. Genetic differentiation between ST22 MRSA inpatient isolates from different hospitals was less than between inpatient isolates from the same hospitals (p ≤ 0.01). Conclusions: There is evidence of frequent ward-based transmission of MRSA brought about by frequent patient admissions to multiple hospitals. Limiting in-ward transmission requires sharing of MRSA-status data between hospitals.

Chlamydia trachomatis(Ct) is the most common infectious cause of blindness and bacterial sexually transmitted infection worldwide. UsingCtwhole genome sequences obtained directly from conjunctival swabs, we studiedCtgenomic diversity and associations betweenCtgenetic polymorphisms with ocular localization and disease severity in a treatment-naïve trachoma-endemic population in Guinea Bissau, West Africa. All sequences fall within the T2 ocular clade phylogenetically. This is consistent with the presence of the characteristic deletion intrpAresulting in a truncated non-functional protein and the ocular tyrosine repeat regions present intarPassociated with ocular tissue localization. We have identified twenty-oneCtnon-synonymous single nucleotide polymorphisms (SNPs) associated with ocular localization, including SNPs withinpmpD(OR=4.07,p*=0.001) andtarP(OR=0.34,p*=0.009). Eight SNPs associated with disease severity were found inyjfH (rlmB)(OR=0.13,p*=0.037),CTA0273(OR=0.12,p*=0.027),trmD(OR=0.12,p*=0.032),CTA0744(OR=0.12,p*=0.041),glgA(OR=0.10,p*=0.026),alaS(OR=0.10,p*=0.032),pmpE(OR=0.08,p*=0.001) and the intergenic regionCTA0744-CTA0745(OR=0.13,p*=0.043). This study demonstrates the extent of genomic diversity within a naturally circulating population of ocularCt, and the first to describe novel genomic associations with disease severity. These findings direct investigation of host-pathogen interactions that may be important in ocularCtpathogenesis and disease transmission.

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 genomewide 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. The genetic diversity and evolutionary plasticity of P. falciparum are major obstacles for malaria elimination. New forms of resistance against antimalarial drugs are continually emerging 1,2 , and new forms of antigenic variation are a critical point of vulnerability for future malaria vaccines. Effective tools are needed to detect evolutionary changes in the parasite population and to monitor the spread of genetic variants that affect malaria control. Here we describe the use of deep sequencing to analyse P. falciparum diversity, using blood samples from patients with malaria. The P. falciparum genome has several unusual features that greatly complicate sequence analysis, such as extreme AT bias, large tracts of nonunique sequence and several large families of intensely polymorphic genes 3 . Our aim was therefore not to determine the entire genome sequence of individual field samples—which would be prohibitively expensive with current technologies—but to define an initial set of single nucleotide polymorphisms (SNPs) distributed across the P. falciparum genome, whose genotype can be ascertained with confidence in parasitized blood samples by deep sequencing. An additional complication in the analysis of P. falciparum genome variation is that the billions of haploid parasites that infect a single individual can be a complex mixture of genetic types. Previous studies 4–8 have largely focused on laboratory-adapted parasite clones, but the within-host diversity of natural infections is of fundamental biological interest. Parasites in the blood replicate asexually, but when they are taken up in the blood meal of an Anopheles mosquito they undergo sexual mating. If the parasites in the blood are of diverse genetic types, this process of sexual mating can generate novel recombinant forms. Deep sequencing provides new ways of investigating within-host diversity and the role of sexual recombination in parasite evolution.

The life cycles of many parasites involve transitions between disparate host species, requiring these parasites to go through multiple developmental stages adapted to each of these specialized niches. Transmission of malaria parasites (Plasmodium spp.) from humans to the mosquito vector requires differentiation from asexual stages replicating within red blood cells into non-dividing male and female gametocytes. Although gametocytes were first described in 1880, our understanding of the molecular mechanisms involved in commitment to gametocyte formation is extremely limited, and disrupting this critical developmental transition remains a long-standing goal. Here we show that expression levels of the DNA-binding protein PfAP2-G correlate strongly with levels of gametocyte formation. Using independent forward and reverse genetics approaches, we demonstrate that PfAP2-G function is essential for parasite sexual differentiation. By combining genome-wide PfAP2-G cognate motif occurrence with global transcriptional changes resulting from PfAP2-G ablation, we identify early gametocyte genes as probable targets of PfAP2-G and show that their regulation by PfAP2-G is critical for their wild-type level expression. In the asexual blood-stage parasites pfap2-g appears to be among a set of epigenetically silenced loci prone to spontaneous activation. Stochastic activation presents a simple mechanism for a low baseline of gametocyte production. Overall, these findings identify PfAP2-G as a master regulator of sexual-stage development in malaria parasites and mark the first discovery of a transcriptional switch controlling a differentiation decision in protozoan parasites.

We describe the haplotypic structure of the interferon regulatory factor-1 (IRF-1) locus in two West African ethnic groups, Fulani and Mossi, that differ in their susceptibility and immune response to Plasmodium falciparum malaria. Both populations showed significant associations between IRF-1 polymorphisms and carriage of P. falciparum infection, with different patterns of association that may reflect their different haplotypic architecture. Genetic variation at this locus does not therefore account for the Fulani-specific resistance to malaria while it could contribute to parasite clearance's ability in populations living in endemic areas. We then conducted a case-control study of three haplotype-tagging single nucleotide polymorphisms (htSNPs) in 370 hospitalised malaria patients (160 severe and 210 uncomplicated) and 410 healthy population controls, all from the Mossi ethnic group. All three htSNPs showed correlation with blood infection levels in malaria patients, and the rs10065633 polymorphism was associated with severe disease (P=0.02). These findings provide the first evidence of the involvement in malaria susceptibility of a specific locus within the 5q31 region, previously shown to be linked with P. falciparum infection levels.

It has been previously shown that there are some interethnic differences in susceptibility to malaria between two sympatric ethnic groups of Mali, the Fulani and the Dogon. The lower susceptibility to Plasmodium falciparum malaria seen in the Fulani has not been fully explained by genetic polymorphisms previously known to be associated with malaria resistance, including haemoglobin S (HbS), haemoglobin C (HbC), alpha-thalassaemia and glucose-6-phosphate dehydrogenase (G6PD) deficiency. Given the observed differences in the distribution of FcγRIIa allotypes among different ethnic groups and with malaria susceptibility that have been reported, we analysed the rs1801274-R131H polymorphism in the FcγRIIa gene in a study of Dogon and Fulani in Mali (n = 939). We confirm that the Fulani have less parasite densities, less parasite prevalence, more spleen enlargement and higher levels of total IgG antibodies (anti-CSP, anti-AMA1, anti-MSP1 and anti-MSP2) and more total IgE (P < 0.05) compared with the Dogon ethnic group. Furthermore, the Fulani exhibit higher frequencies of the blood group O (56.5%) compared with the Dogon (43.5%) (P < 0.001). With regard to the FcγRIIa polymorphism and allele frequency, the Fulani group have a higher frequency of the H allele (Fulani 0.474, Dogon 0.341, P < 0.0001), which was associated with greater total IgE production (P = 0.004). Our findings show that the FcγRIIa polymorphism might have an implication in the relative protection seen in the Fulani tribe, with confirmatory studies required in other malaria endemic settings.

Characterizing the drug resistance mutations that have evolved in Mycobacterium tuberculosis (Mtb), has important implications for control of tuberculosis (TB) disease, through more accurate and timely use of therapy. Whole genome sequencing of Mtb can assist this characterization by providing insights into loci and specific mutations underlying drug resistance and the transmission success that enables their spread. We hypothesised that genetic variation outside of known resistance-conferring mutations might give additional information concerning drug resistance and fitness. Firstly, we explored the effect of lineage on the identification of drug resistance associations, applying novel lineage level genome-wide association study (GWAS) and convergence-based (PhyC) methods to drug resistance phenotypes of a global dataset of Mtb lineages 2 and 4. We identified known drug resistance variants and novel associations, uniquely identifying associations for lineage-specific GWAS analyses and reporting 17 novel associations between antimicrobial resistance phenotypes and Mtb genomic variants, demonstrating the utility of lineage-specific GWAS. To further examine the genomic basis of extensively drug resistant (XDR)-TB, we next applied the GWAS and PhyC techniques to a global dataset of 18,255 Mtb isolates. Through GWAS we identified 20 loci in novel associations within highly drug-resistant Mtb strains. Cluster-based GWAS and a lack of overlap with associations identified through convergent-evolution-based analyses confirmed that many such associations have been driven by transmission in outbreaks of XDR-TB. We then investigated the feasibility of applying a learning classifier system to this dataset to predict rifampicin resistance and discover candidate loci for novel involvement, finally enabling a sensitivity of 93.7% and a specificity of 94.8% of rifampicin resistance prediction. Finally, we applied this methodology to the XDR phenotype in lineages 2 and 4 of a global dataset (n=13,270), achieving high accuracy of prediction and identifying a number of candidate loci for involvement in XDR, including candidates for epistasis.

Malaria is a major threat to human health, causing over 300 million clinical cases and approximately ~500,000 deaths per year. Countries attempting malaria elimination are increasingly concerned with identifying pockets of transmission and outbreaks arising from imported cases, and there is a need to establish molecular barcodes for implementation in the field. The genetic diversity and non-recombining properties of mitochondrial and apicoplast sequence can be powerfully exploited for geographic genetic profiling of P. falciparum malaria at an inter-continent level. However, this approach provides limited insights for assessing drug resistance, intra-regional geographical differentiation, and ignores malaria caused by other Plasmodium spp. (P. vivax and P. knowlesi). To overcome these limitations, this project proposes to study the genomic diversity found in the nuclear and organellar genomes of the Plasmodium species causing human malaria and establish robust ways to create SNP barcodes. In this study, an assessment of the current libraries of genomic sequence data across the species P. falciparum, P. vivax and P. knowlesi was performed and using a range of bioinformatics approaches the genetic diversity in the different populations was assessed. For this, a new high-quality reference for the A1-H.1 P. knowlesi strain was generated and its methylome was characterized. Using this reference, the first evidence of genetic exchange events between the three subpopulations of P. knowlesi was found in Malaysia. Furthermore, a study of the structural and genetic diversity found in the hypervariable vaccine candidate var2csa gene in P. falciparum and its potential geographical signal associated with Malaria in Pregnancy (MiP) were performed. Finally, we accomplished a genetic diversity study of global P. vivax isolates and the insights obtained from this analysis allowed the development of a 71 SNP barcode to predict the geographical origin of P. vivax isolates. The obtained barcode were tested using prospectively and retrospectively collected datasets, particularly from endemic settings with complex mixed infections and near-elimination settings. The identification of SNP barcodes using this methodology can inform future rapid diagnostics and promote the application of field-based sequencing.

Similarity between two individuals in the combination of genetic markers along their chromosomes indicates shared ancestry and can be used to identify historical connections between different population groups due to admixture. We use a genome-wide, haplotype-based, analysis to characterise the structure of genetic diversity and gene-flow in a collection of 48 sub-Saharan African groups. We show that coastal populations experienced an influx of Eurasian haplotypes over the last 7000 years, and that Eastern and Southern Niger-Congo speaking groups share ancestry with Central West Africans as a result of recent population expansions. In fact, most sub-Saharan populations share ancestry with groups from outside of their current geographic region as a result of gene-flow within the last 4000 years. Our in-depth analysis provides insight into haplotype sharing across different ethno-linguistic groups and the recent movement of alleles into new environments, both of which are relevant to studies of genetic epidemiology. DOI: http://dx.doi.org/10.7554/eLife.15266.001, eLife digest Our genomes contain a record of historical events. This is because when groups of people are separated for generations, the DNA sequence in the two groups’ genomes will change in different ways. Looking at the differences in the genomes of people from the same population can help researchers to understand and reconstruct the historical interactions that brought their ancestors together. The mixing of two populations that were previously separate is known as admixture. Africa as a continent has few written records of its history. This means that it is somewhat unknown which important movements of people in the past generated the populations found in modern-day Africa. Busby et al. have now attempted to use DNA to look into this and reconstruct the last 4000 years of genetic history in African populations. As has been shown in other regions of the world, the new analysis showed that all African populations are the result of historical admixture events. However, Busby et al. could characterize these events to unprecedented level of detail. For example, multiple ethnic groups from The Gambia and Mali all show signs of sharing the same set of ancestors from West Africa, Europe and Asia who mixed around 2000 years ago. Evidence of a migration of people from Central West Africa, known as the Bantu expansion, could also be detected, and was shown to carry genes to the south and east. An important next step will be to now look at the consequences of the observed gene-flow, and ask if it has contributed to spreading beneficial, or detrimental, mutations around Africa. DOI: http://dx.doi.org/10.7554/eLife.15266.002

Austism is a common, severe and highly heritable neurodevelopmental disorder. The International Molecular Genetic Study of Autism Consortium (IMGSAC) genome screen for linkage in affected sib-pair families identified a chromosome 7 q susceptibility locus (AUTS1), that has subsequently shown evidence of increased sharing in several independent multiplex samples and in two meta-anlyses. Taking into account the location of the MET gene under this linkage peak, and the fact that it has recently been reported to be associated with autism, the gene was further analyzed as a promising autism candidate. The gene encodes a transmembrane receptor tyrosine kinase of the hepatocyte growth factor/scatter factor (HGF/SF). MET is best known as an oncogene, but its signalling also participates in immune function, peripheral organ development and repair, and the development of the cerebral cortex and cerebellum (all of which have been observed earlier as being disregulated in individuals with autism). Here we present a family-based association analysis covering the entire MET locus. Significant results were obtained in both single locus and haplotype approaches with a single nucleotide polymorphism in intron 1 (rs38845, P < 0.004) and with one intronic haplotype (AAGTG, P < 0.009) in 325 multiplex IMGSAC families and 10 IMGSAC trios. Although these results failed to replicate in an independent sample of 82 Italian trios, the association itself was confirmed by a case-control analysis performed using the Italian cohort (P < 0.02). The previously reported positive association of rs1858830 failed to replicate in this study. Overall, our findings provide further evidence that MET may play a role in autism susceptibility.

Tuberculosis (TB) caused by bacteria of the Mycobacterium- tuberculosis complex \ud (MTBC) is the second major cause of death from an infectious disease worldwide. \ud Recent advances in DNA sequencing are leading to the ability to generate whole \ud genome information of clinical isolates of MTBC. The objectives of this work include \ud developing bioinformatic tools for processing and making accessible MTBC genomic \ud data, as well as the identification of informative genetic markers, both strainOspecific \ud and associated with drug resistance (DR), to barcode MTBC isolates in research and \ud clinical settings. \ud SpolPred software was developed to accurately predict the spoligotype from raw \ud sequence reads, and used to bridge the gap between classical genotyping and highO\ud throughput sequencing. A genome variation discovery pipeline was implemented to \ud derive genomic polymorphisms from MTBC raw sequence data. This pipeline was \ud applied to >1,500 publicly available isolates and the characterised genomic variation \ud hosted in PolyTB, a webObased tool where genetic variants can be investigated using a \ud genome browser, a world map showing their global allele distribution, and an \ud additional phylogenetic view. An extensive repertoire of strainOspecific mutations was \ud identified, of which a subset was proposed to accurately discriminate known MTBC \ud circulating strains. A curated list of DR associated mutations was compiled from the \ud literature and their diagnostic accuracy for predicting phenotypic resistance assessed. \ud In addition, potentially novel genes involved in DR were discovered by applying \ud genomeOwide association approaches to a global population of more than 2,500 MTBC \ud strains. Whole genome sequencing (WGS) promises to be transformative for the practice of \ud clinical microbiology, and the rapidly falling cost and turnaround time mean that this \ud will become a viable technology in clinical settings. In this new paradigm, the \ud presented work will facilitate the transition to and applications of WGS in clinical \ud settings as an important tool for TB control.

Human tuberculosis disease (TB) is caused by bacteria within the Mycobacterium tuberculosis complex, including M. tuberculosis (Mtb). Genetic variation within the pathogen can lead to drug resistance, affect virulence and transmissibility. I have analysed Mtb whole genome sequence data to improve the understanding of global genetic variation, and the resulting insights could ultimately assist the development of TB control measures.\ud Whole genome sequencing platforms are being used to infer drug resistance profiles, and thereby could assist clinical management. I investigated the reproducibility of sequence data from two platforms (Illumina MiSeq, Ion Torrent PGM™) and two rapid analytic pipelines (TBProfiler, Mykrobe predictor). DNA replicates from the reference strain (H37Rv) and 10 drug-resistant strains were sequenced, and inferred drug resistance genotypes were compared to drug susceptibility testing phenotypes.\ud Genome-wide association study (GWAS) can be used to detect mutations associated with Mtb drug resistance. A first GWAS (n=127) attempted to identify mutations associated with minimum inhibitory concentrations for first-line anti-tuberculosis drugs. A second GWAS was applied to a large global set (n>6400) to identify mutations associated with first- and secondline drug resistance.\ud M. aurum is an environmental mycobacteria that has been proposed as a model for the development of anti-TB drugs. I have assembled and annotated its draft genome, and identified copy number variants in known drug resistance targets.\ud Approximately 10% of the Mtb genome consists of two gene families (pe/ppe) that are poorly characterised, and are hypothesised to be important virulence factors. Using a de novo assembly approach, I characterised these genes and their diversity across a global collection of clinical isolates with high depth short-read sequence data (n=518). A follow-up study using a long-read sequence technology (n=18, diverse stain types) confirmed the findings. This work also generated new annotated reference genomes and characterised methylation sites, which may affect transmissibility, pathogenicity and virulence.\ud A future direction of the TB genomics field is to identify genetic check points in host-pathogen\ud interactions using both human and Mtb genotypes. I analysed the genomes of ~720 TB case–Mtb pairs and identified susceptibility markers, which are promising targets for future control measures.