Faculty Bibliography

BACKGROUND: Amplicon pyrosequencing targets a known genetic region and thus inherently produces reads highly anticipated to have certain features, such as conserved nucleotide sequence, and in the case of protein coding DNA, an open reading frame. Pyrosequencing errors, consisting mainly of nucleotide insertions and deletions, are on the other hand likely to disrupt open reading frames. Such an inverse relationship between errors and expectation based on prior knowledge can be used advantageously to guide the process known as basecalling, i.e. the inference of nucleotide sequence from raw sequencing data. RESULTS: The new basecalling method described here, named Multipass, implements a probabilistic framework for working with the raw flowgrams obtained by pyrosequencing. For each sequence variant Multipass calculates the likelihood and nucleotide sequence of several most likely sequences given the flowgram data. This probabilistic approach enables integration of basecalling into a larger model where other parameters can be incorporated, such as the likelihood for observing a full-length open reading frame at the targeted region. We apply the method to 454 amplicon pyrosequencing data obtained from a malaria virulence gene family, where Multipass generates 20 % more error-free sequences than current state of the art methods, and provides sequence characteristics that allow generation of a set of high confidence error-free sequences. CONCLUSIONS: This novel method can be used to increase accuracy of existing and future amplicon sequencing data, particularly where extensive prior knowledge is available about the obtained sequences, for example in analysis of the immunoglobulin VDJ region where Multipass can be combined with a model for the known recombining germline genes. Multipass is available for Roche 454 data at http://www.cbs.dtu.dk/services/MultiPass-1.0 , and the concept can potentially be implemented for other sequencing technologies as well.

BACKGROUND: Recent studies described an increase in acute kidney injury when high dose gentamicin was included in perioperative prophylaxis for orthopedic surgeries. To this effect, we compared the rate of nephrotoxicity for selected orthopedic surgeries where gentamicin was included (Gentamicin Group) to those where it was not included (Control Group) for perioperative prophylaxis and evaluated risk factors for nephrotoxicity. METHODS: Spine, hip and knee surgeries performed between April 2011 and December 2013 were reviewed retrospectively. Gentamicin was given to eligible patients based on age, weight and Creatinine Clearance. Nephrotoxicity was assessed using Risk, Injury, Failure, Loss, End-stage kidney disease (RIFLE) criteria. RESULTS: Among selected surgeries (N = 1590 in Gentamicin Group: hip = 926, spine = 600, knee = 64; N = 2587 in CONTROL GROUP: hip = 980, spine = 902, knee = 705), patients' body weight, serum creatinine, comorbidities and surgery duration were similar in Gentamicin Group and CONTROL GROUP. Gentamicin median dose was 4.5 mg/kg of dosing weight. Nephrotoxicity rate was 2.5% in Gentamicin Group and 1.8% in CONTROL GROUP, p = 0.17. Most cases of nephrotoxicity were Risk category by RIFLE criteria (67% in Gentamicin Group and 72% in CONTROL GROUP, p = 0.49). In logistic regression, risk factors for nephrotoxicity were hospital stay >1 day prior to surgery (odds ratio = 8.1; 95% confidence interval = 2.25-28.97, p = 0.001), knee or hip surgery (odds ratio = 4.7; 95% confidence interval = 2.9-9.48, p = 0.0005) and diabetes (odds ratio = 1.95; 95% confidence interval = 1.13-3.35, p = 0.016). Receipt of gentamicin was not an independent predictor of nephrotoxicity (odds ratio = 1.5; 95% confidence interval = 0.97-2.35, p = 0.07). CONCLUSION: In this cohort, rate of nephrotoxicity was similar between Gentamicin Group and Control Group. Single high dose gentamicin is a safe and acceptable option for perioperative prophylaxis in eligible patients undergoing orthopedic surgeries.

Piperacillin-tazobactam (PTZ) is frequently used as empirical and targeted therapy for Gram-negative sepsis. Time-dependent killing properties of PTZ support the use of extended-infusion (EI) dosing; however, studies have shown inconsistent benefits of EI PTZ treatment on clinical outcomes. We performed a retrospective cohort study of adult patients who received EI PTZ treatment and historical controls who received standard-infusion (SI) PTZ treatment for presumed sepsis syndromes. Data on mortality rates, clinical outcomes, length of stay (LOS), and disease severity were obtained. A total of 843 patients (662 with EI treatment and 181 with SI treatment) were available for analysis. Baseline characteristics of the two groups were similar, except for fewer female patients receiving EI treatment. No significant differences between the EI and SI groups in inpatient mortality rates (10.9% versus 13.8%; P = 0.282), overall LOS (10 versus 12 days; P = 0.171), intensive care unit (ICU) LOS (7 versus 6 days; P = 0.061), or clinical failure rates (18.4% versus 19.9%; P = 0.756) were observed. However, the duration of PTZ therapy was shorter in the EI group (5 versus 6 days; P < 0.001). Among ICU patients, no significant differences in outcomes between the EI and SI groups were observed. Patients with urinary or intra-abdominal infections had lower mortality and clinical failure rates when receiving EI PTZ treatment. We did not observe significant differences in inpatient mortality rates, overall LOS, ICU LOS, or clinical failure rates between patients receiving EI PTZ treatment and patients receiving SI PTZ treatment. Patients receiving EI PTZ treatment had a shorter duration of PTZ therapy than did patients receiving SI treatment, and EI dosing may provide cost savings to hospitals.

BACKGROUND: The var genes of the human malaria parasite Plasmodium falciparum are highly polymorphic loci coding for the erythrocyte membrane proteins 1 (PfEMP1), which are responsible for the cytoaherence of P. falciparum infected red blood cells to the human vasculature. Cytoadhesion, coupled with differential expression of var genes, contributes to virulence and allows the parasite to establish chronic infections by evading detection from the host's immune system. Although studying genetic diversity is a major focus of recent work on the var genes, little is known about the gene family's origin and evolutionary history. RESULTS: Using a novel hidden Markov model-based approach and var sequences assembled from additional isolates and species, we are able to reveal elements of both the early evolution of the var genes as well as recent diversifying events. We compare sequences of the var gene DBLalpha domains from divergent isolates of P. falciparum (3D7 and HB3), and a closely-related species, Plasmodium reichenowi. We find that the gene family is equally large in P. reichenowi and P. falciparum -- with a minimum of 51 var genes in the P. reichenowi genome (compared to 61 in 3D7 and a minimum of 48 in HB3). In addition, we are able to define large, continuous blocks of homologous sequence among P. falciparum and P. reichenowi var gene DBLalpha domains. These results reveal that the contemporary structure of the var gene family was present before the divergence of P. falciparum and P. reichenowi, estimated to be between 2.5 to 6 million years ago. We also reveal that recombination has played an important and traceable role in both the establishment, and the maintenance, of diversity in the sequences. CONCLUSIONS: Despite the remarkable diversity and rapid evolution found in these loci within and among P. falciparum populations, the basic structure of these domains and the gene family is surprisingly old and stable. Revealing a common structure as well as conserved sequence among two species also has implications for developing new primate-parasite models for studying the pathology and immunology of falciparum malaria, and for studying the population genetics of var genes and associated virulence phenotypes.

The coexistence of multiple independently circulating strains in pathogen populations that undergo sexual recombination is a central question of epidemiology with profound implications for control. An agent-based model is developed that extends earlier 'strain theory' by addressing the var gene family of Plasmodium falciparum. The model explicitly considers the extensive diversity of multi-copy genes that undergo antigenic variation via sequential, mutually exclusive expression. It tracks the dynamics of all unique var repertoires in a population of hosts, and shows that even under high levels of sexual recombination, strain competition mediated through cross-immunity structures the parasite population into a subset of coexisting dominant repertoires of var genes whose degree of antigenic overlap depends on transmission intensity. Empirical comparison of patterns of genetic variation at antigenic and neutral sites supports this role for immune selection in structuring parasite diversity.DOI:http://dx.doi.org/10.7554/eLife.00093.001.

BACKGROUND: The reservoir of Plasmodium infection in humans has traditionally been defined by blood slide positivity. This study was designed to characterize the local reservoir of infection in relation to the diverse var genes that encode the major surface antigen of Plasmodium falciparum blood stages and underlie the parasite's ability to establish chronic infection and transmit from human to mosquito. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the molecular epidemiology of the var multigene family at local sites in Gabon, Senegal and Kenya which differ in parasite prevalence and transmission intensity. 1839 distinct var gene types were defined by sequencing DBLalpha domains in the three sites. Only 76 (4.1%) var types were found in more than one population indicating spatial heterogeneity in var types across the African continent. The majority of var types appeared only once in the population sample. Non-parametric statistical estimators predict in each population at minimum five to seven thousand distinct var types. Similar diversity of var types was seen in sites with different parasite prevalences. CONCLUSIONS/SIGNIFICANCE: Var population genomics provides new insights into the epidemiology of P. falciparum in Africa where malaria has never been conquered. In particular, we have described the extensive reservoir of infection in local African sites and discovered a unique var population structure that can facilitate superinfection through minimal overlap in var repertoires among parasite genomes. Our findings show that var typing as a molecular surveillance system defines the extent of genetic complexity in the reservoir of infection to complement measures of malaria prevalence. The observed small scale spatial diversity of var genes suggests that var genetics could greatly inform current malaria mapping approaches and predict complex malaria population dynamics due to the import of var types to areas where no widespread pre-existing immunity in the population exists