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

Activation of minority-variant Plasmodium vivax hypnozoites following artesunate + amodiaquine treatment in a 23-year old man with relapsing malaria in Antananarivo, Madagascar

In endemic areas, Plasmodium vivax relapses are difficult to distinguish from new infections. Genotyping of patients who experience relapse after returning to a malaria-free area can be used to explore the nature of hypnozoite activation and relapse. This paper describes a person who developed P. vivax malaria for the first time after travelling to Boriziny in the malaria endemic coastal area of Madagascar, then suffered two P. vivax relapses 11 weeks and 21 weeks later despite remaining in Antananarivo in the malaria-free central highlands area. He was treated with the combination artesunate + amodiaquine according to the national malaria policy in Madagascar. Genotyping by PCR-RFLP at pvmsp-3α as well as pvmsp1 heteroduplex tracking assay (HTA) showed the same dominant genotype at each relapse. Multiple recurring minority variants were also detected at each relapse, highlighting the propensity for multiple hypnozoite clones to activate simultaneously to cause relapse

Can pharmacogenomics improve malaria drug policy?

Coordinated global efforts to prevent and control malaria have been a tour-de-force for public health, but success appears to have reached a plateau in many parts of the world. While this is a multifaceted problem, policy strategies have largely ignored genetic variations in humans as a factor that influences both selection and dosing of antimalarial drugs. This includes attempts to decrease toxicity, increase effectiveness and reduce the development of drug resistance, thereby lowering health care costs. We review the potential hurdles to developing and implementing pharmacogenetic-guided policies at a national or regional scale for the treatment of uncomplicated falciparum malaria. We also consider current knowledge on some component drugs of artemisinin combination therapies and ways to increase our understanding of host genetics, with the goal of guiding policy decisions for drug selection

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

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

Short Report: Detection of the Dihydrofolate Reductase–164L Mutation in Plasmodium falciparum Infections from Malawi by Heteroduplex Tracking Assay

Standard polymerase chain reaction methods often cannot detect drug-resistance mutations in Plasmodium falciparum infections if the mutation is present in ≤ 20% of the parasites. A heteroduplex tracking assay was developed that can detect dihydrofolate reductase 164-L mutations in variants representing 1% of the parasites in an individual host. Using this assay, we confirmed the presence of the mutation in P. falciparum infections in Malawi

Longevity of Genotype-Specific Immune Responses to Plasmodium falciparum Merozoite Surface Protein 1 in Kenyan Children from Regions of Different Malaria Transmission Intensity

Naturally acquired immunity to Plasmodium falciparum presents a changing landscape as malaria control programs and vaccine initiatives are implemented. Determining which immunologic indicators remain surrogates of past infection, as opposed to mediators of protection, led us to compare stability of immune responses across regions with divergent malaria transmission intensities. A repeat cross-sectional study of Kenyan children from a malaria-holoendemic area and an epidemic-prone area was used to examine longitudinal antibody and interferon-gamma (IFN-gamma) responses to the 3D7 and FVO variants of merozoite surface protein 1 (MSP1). Antibodies to MSP1 were common in both study populations and did not significantly wane over a 21-month time period. IFN-gamma responses were less frequent and rapidly disappeared in children after a prolonged period of no malaria transmission. Antibody and IFN-gamma responses rarely correlated with each other; however, MSP1-specific IFN-gamma response correlated with lack of concurrent P. falciparum parasitemia of the same genotype, though only statistically significantly in the malaria-holoendemic region (odds ratio = 0.31, 95% confidence interval = 0.12-0.84). This study affirms that antimalarial antibodies are informative for evaluation of history of malaria exposure within individuals, whereas cell-mediated immunity, though short lived under natural exposure conditions, might provide an assessment of recent infection and protection from parasitemia

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

Acute Chagas Disease in a Returning Traveler

Acute Chagas disease is rarely recognized, and the risk for acquiring the disease is undefined in travelers to Central America. We describe a case of acute Chagas disease in a traveler to Costa Rica and highlight the need for increased awareness of this infection in travelers to Chagas-endemic areas

Short Report: Novel dhps and pfcrt Polymorphisms in Plasmodium falciparum Detected by Heteroduplex Tracking Assay

Several single nucleotide polymorphisms (SNPs) have been linked to antimalarial drug resistance in Plasmodium falciparum . However, standard polymerase chain reaction (PCR) methods to detect these polymorphisms are unable to detect SNPs in variants representing < 20% of the parasites in a mixed infection, nor can they detect polymorphisms at nearby loci. Here we use heteroduplex tracking assays (HTAs) to analyze dhps540 mutations in 96 samples from Peru and pfcrt76 mutations in 70 samples from China. All samples had been previously analyzed by standard PCR. We detected drug-resistant minority variants and two novel non-synonymous pfcrt mutations in China. In Peru, we found no drug-resistant minority variants and a synonymous mutation in dhps. Thus, even in regions of low malaria transmission, HTA assays are more informative than PCR with agarose gel electrophoresis