Dissecting the Effects of Selective Pressures on the Genomes of Co-endemic Plasmodium vivax and Plasmodium falciparum in Cambodia

Each year, the malaria parasites Plasmodium vivax and Plasmodium falciparum together infect hundreds of millions of people and cause hundreds of thousands of deaths. In Cambodia, these two parasites are co-endemic, transmitted by the same vectors, and cause disease in the same populations. Recently, pointed efforts have been made in western Cambodia to eliminate malaria. This selective pressure provides a natural experiment that enables direct comparison of P. vivax and P. falciparum populations. Here, we explore the genetic similarities and differences between co-endemic P. vivax and P. falciparum from Cambodia through three questions to be answered. First, we conducted a pilot study in which we deep sequenced two important antigens - pvmsp1 and pvcsp - from 48 P. vivax clinical isolates and explored the contrasting population genetics of these two antigens. We discovered that, in population genetic studies, marker choice has a profound effect on study outcomes, and that P. vivax and P. falciparum orthologs can host very different signatures of selection. Next, through whole-genome sequencing of 80 P. falciparum isolates, we found that population substructuring associates with artemisinin-combination therapy partner-drug in vitro IC50 and with clinical outcomes. This finding suggests that the P. falciparum population has in part responded to selective pressures with complex demographic changes. Finally, we whole-genome sequenced 70 sympatric P. vivax isolates. We found that, in spite of control efforts and in contrast to P. falciparum, the genetic diversity within the P. vivax population remains high and has been more rapidly expanding. We identified a minority of orthologous loci that have opposite signatures of selection in P. vivax and P. falciparum. Surprisingly, we found evidence of several strong and recent selective sweeps within the P. vivax population at transcriptional regulatory loci. This finding suggests that P. vivax may rely on a nuanced response to selective pressure, modulating transcript levels as a means to maintain population resilience.Doctor of Philosoph

SeekDeep: single-base resolution de novo clustering for amplicon deep sequencing

PCR amplicon deep sequencing continues to transform the investigation of genetic diversity in viral, bacterial, and eukaryotic populations. In eukaryotic populations such as Plasmodium falciparum infections, it is important to discriminate sequences differing by a single nucleotide polymorphism. In bacterial populations, single-base resolution can provide improved resolution towards species and strains. Here, we introduce the SeekDeep suite built around the qluster algorithm, which is capable of accurately building de novo clusters representing true, biological local haplotypes differing by just a single base. It outperforms current software, particularly at low frequencies and at low input read depths, whether resolving single-base differences or traditional OTUs. SeekDeep is open source and works with all major sequencing technologies, making it broadly useful in a wide variety of applications of amplicon deep sequencing to extract accurate and maximal biologic information

Haemoglobinopathies and the clinical epidemiology of malaria: a systematic review and meta-analysis

Haemoglobinopathies variously reduce the risk of developing malaria syndromes. Quantifying these relationships may strengthen the foundation for translational studies of malaria pathogenesis and immunity

skelesim : an extensible, general framework for population genetic simulation in R

Simulations are a key tool in molecular ecology for inference and forecasting, as well as for evaluating new methods. Due to growing computational power and a diversity of software with different capabilities, simulations are becoming increasingly powerful and useful. However, the widespread use of simulations by geneticists and ecologists is hindered by difficulties in understanding these softwares’ complex capabilities, composing code and input files, a daunting bioinformatics barrier, and a steep conceptual learning curve. skeleSim (an R package) guides users in choosing appropriate simulations, setting parameters, calculating genetic summary statistics, and organizing data output, in a reproducible pipeline within the R environment. skeleSim is designed to be an extensible framework that can ‘wrap’ around any simulation software (inside or outside the R environment) and be extended to calculate and graph any genetic summary statistics. Currently, skeleSim implements coalescent and forward-time models available in the fastsimcoal2 and rmetasim simulation engines to produce null distributions for multiple population genetic statistics and marker types, under a variety of demographic conditions. skeleSim is intended to make simulations easier while still allowing full model complexity to ensure that simulations play a fundamental role in molecular ecology investigations. skeleSim can also serve as a teaching tool: demonstrating the outcomes of stochastic population genetic processes; teaching general concepts of simulations; and providing an introduction to the R environment with a user-friendly graphical user interface (using shiny)

Next-Generation Sequencing and Comparative Analysis of Sequential Outbreaks Caused by Multidrug-Resistant Acinetobacter baumannii at a Large Academic Burn Center

Next-generation sequencing (NGS) analysis has emerged as a promising molecular epidemiological method for investigating health care-associated outbreaks. Here, we used NGS to investigate a 3-year outbreak of multidrug-resistant Acinetobacter baumannii (MDRAB) at a large academic burn center. A reference genome from the index case was generated using de novo assembly of PacBio reads. Forty-six MDRAB isolates were analyzed by pulsed-field gel electrophoresis (PFGE) and sequenced using an Illumina platform. After mapping to the index case reference genome, four samples were excluded due to low coverage, leaving 42 samples for further analysis. Multilocus sequence types (MLST) and the presence of acquired resistance genes were also determined from the sequencing data. A transmission network was inferred from genomic and epidemiological data using a Bayesian framework. Based on single-nucleotide variant (SNV) differences, this MDRAB outbreak represented three sequential outbreaks caused by distinct clones. The first and second outbreaks were caused by sequence type 2 (ST2), while the third outbreak was caused by ST79. For the second outbreak, the MLST and PFGE results were discordant. However, NGS-based SNV typing detected a recombination event and consequently enabled a more accurate phylogenetic analysis. The distribution of resistance genes varied among the three outbreaks. The first- and second-outbreak strains possessed a bla OXA-23-like group, while the third-outbreak strains harbored a bla OXA-40-like group. NGS-based analysis demonstrated the superior resolution of outbreak transmission networks for MDRAB and provided insight into the mechanisms of strain diversification between sequential outbreaks through recombination

A prolonged outbreak of KPC-3-producing Enterobacter cloacae and Klebsiella pneumoniae driven by multiple mechanisms of resistance transmission at a large academic burn center

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter cloacae have been recently recognized in the United States. Whole-genome sequencing (WGS) has become a useful tool for analysis of outbreaks and for determining transmission networks of multidrug-resistant organisms in healthcare settings, including carbapenem-resistant Enterobacteriaceae (CRE). We experienced a prolonged outbreak of CRE of E. cloacae and K. pneumoniae over a three-year period at a large academic burn center despite rigorous infection control measures. To understand the molecular mechanisms that sustained this outbreak, we investigated the CRE outbreak isolates using WGS. Twenty-two clinical isolates of CRE, including E. cloacae (N=15) and K. pneumoniae (N=7), were sequenced and analyzed genetically. WGS revealed that this outbreak, which seemed epidemiologically unlinked, was in fact genetically linked over a prolonged period. Multiple mechanisms were found to account for the ongoing outbreak of KPC-3-producing E. cloacae and K. pneumoniae . This outbreak was primarily maintained by a clonal expansion of E. cloacae ST114 with distribution of multiple resistance determinants. Plasmid and transposon analysis suggested that the majority of bla KPC-3 was transmitted via an identical Tn 4401 b element on part of a common plasmid. WGS analysis demonstrated complex transmission dynamics within the burn center at levels of strain and/or plasmid in association with transposon, highlighting the versatility of KPC-producing Enterobacteriaceae in their ability to utilize multiple modes to resistance-gene propagation

Longitudinal Pooled Deep Sequencing of the Plasmodium vivax K12 Kelch Gene in Cambodia Reveals a Lack of Selection by Artemisinin

The emergence of artemisinin resistance among Plasmodium falciparum in the Greater Mekong subregion threatens malaria control interventions and is associated with multiple unique mutations in K13 (PF3D7_1343700). The aim of this study was to survey Cambodian Plasmodium vivax for mutations in the K13 ortholog (K12, PVX_083080) that might similarly confer artemisinin resistance. Extracted DNA from Cambodian isolates collected between 2009 and 2012 was pooled by province and year and submitted for next-generation sequencing. Single-nucleotide polymorphisms (SNPs) were identified using a pile-up approach that detected minority SNPs. Among the 14 pools, we found six unique SNPs, including three nonsynonymous SNPs, across six codons in K12. However, none of the SNPs were orthologous to artemisinin resistance–conferring mutations in PF3D7_1343700, and nonsynonymous changes did not persist through time within populations. These results suggest a lack of selection in the P. vivax population in Cambodia due to artemisinin drug pressure

Genomic Analysis of Multidrug-Resistant Escherichia coli from North Carolina Community Hospitals: Ongoing Circulation of CTX-M-Producing ST131- H 30Rx and ST131- H 30R1 Strains

ABSTRACT Escherichia coli sequence type 131 (ST131) predominates globally among multidrug-resistant (MDR) E. coli strains. We used whole-genome sequencing (WGS) to investigate 63 MDR E. coli isolates from 7 North Carolina community hospitals (2010 to 2015). Of these, 39 (62%) represented ST131, including 37 (95%) from the ST131- H 30R subclone: 10 (27%) from its H 30R1 subset and 27 (69%) from its H 30Rx subset. ST131 core genomes differed by a median of 15 (range, 0 to 490) single-nucleotide variants (SNVs) overall versus only 7 within H 30R1 (range, 3 to 12 SNVs) and 11 within H 30Rx (range, 0 to 21). The four isolates with identical core genomes were all H 30Rx. Epidemiological and clinical characteristics did not vary significantly by strain type, but many patients with MDR E. coli or H 30Rx infection were critically ill and had poor outcomes. H 30Rx isolates characteristically exhibited fluoroquinolone resistance and CTX-M-15 production, had a high prevalence of trimethoprim-sulfamethoxazole resistance (89%), sul1 (89%), and dfrA17 (85%), and were enriched for specific virulence traits, and all qualified as extraintestinal pathogenic E. coli . The high overall prevalence of CTX-M-15 appeared to be possibly attributable to its association with the ST131- H 30Rx subclone and IncF[F2:A1:B−] plasmids. Some phylogenetically clustered non-ST131 MDR E. coli isolates also had distinctive serotypes/ fimH types, fluoroquinolone mutations, CTX-M variants, and IncF types. Thus, WGS analysis of our community hospital source MDR E. coli isolates suggested ongoing circulation and differentiation of E. coli ST131 subclones, with clonal segregation of CTX-M variants, other resistance genes, Inc-type plasmids, and virulence genes

Pooled Deep Sequencing of Plasmodium falciparum Isolates: An Efficient and Scalable Tool to Quantify Prevailing Malaria Drug-Resistance Genotypes

Molecular surveillance for drug-resistant malaria parasites requires reliable, timely, and scalable methods. These data may be efficiently produced by genotyping parasite populations using second-generation sequencing (SGS). We designed and validated a SGS protocol to quantify mutant allele frequencies in the Plasmodium falciparum genes dhfr and dhps in mixed isolates. We applied this new protocol to field isolates from children and compared it to standard genotyping using Sanger sequencing. The SGS protocol accurately quantified dhfr and dhps allele frequencies in a mixture of parasite strains. Using SGS of DNA that was extracted and then pooled from individual isolates, we estimated mutant allele frequencies that were closely correlated to those estimated by Sanger sequencing (correlations, >0.98). The SGS protocol obviated most molecular steps in conventional methods and is cost saving for parasite populations >50. This SGS genotyping method efficiently and reproducibly estimates parasite allele frequencies within populations of P. falciparum for molecular epidemiologic studies

Use of Oropharyngeal Washes to Diagnose and Genotype Pneumocystis jirovecii

Pneumocystis is an important opportunistic pathogen in immunocompromised patients. Molecular epidemiology studies are needed to understand transmission. This article evaluates non-invasive sampling and a new strain typing tool, both of which could be used for this purpose.Pneumocystis jirovecii is a symbiotic respiratory fungus that presents in 2 clinical forms: pneumonia in immunocompromised patients or colonization, defined by the presence of the organism without associated clinical symptoms. Currently, diagnosis requires invasive bronchoscopy, which may not be available in some settings and is inappropriate for detecting colonization in healthy individuals. Noninvasive diagnostic techniques and molecular strain typing tools that can be used on these samples are critical for conducting studies to better understand transmission. We evaluated 2 real-time polymerase chain reaction (PCR) assays targeting dihydropteroate synthase and the major surface glycoprotein for detection in 77 oropharyngeal washes (OPWs) from 43 symptomatic human immunodeficiency virus-infected patients who underwent bronchoscopy. We also evaluated the ability of a new microsatellite (MS) genotyping panel to strain type infections from these samples. Each PCR used individually provided a high sensitivity (>80%) for detection of pneumonia but a modest specificity (<70%). When used in combination, specificity was increased to 100% with a drop in sensitivity (74%). Concentration of organisms by PCR in the OPW tended to be lower in colonized individuals compared with those with pneumonia, but differences in concentration could not clearly define colonization in symptomatic individuals. Oropharyngeal wash samples were genotyped using 6 MSs with ≥4 alleles successfully genotyped in the majority of colonized patients and ≥5 alleles in patients with pneumonia. The MS profile was consistent over time within patients with serial OPWs analyzed. Microsatellite genotyping on noninvasive samples may aid in studying the molecular epidemiology of this pathogen without requiring invasive diagnostic techniques