Publications

In Press
S Leavitt, RS Lee, P. Sebastiani, CR Horsburgh, HE Jenkins, and L White. In Press. “Estimating the Relative Probability of Direct Transmission between Infectious Disease Patients.” International Journal of Epidemiology.
K Břinda, A Callendrello, L Cowley, T Charalampous, RS Lee, DR MacFadden, G Kucherov, J O'Grady, M Baym, and WP Hanage. In Press. “Rapid heuristic inference of antibiotic resistance and susceptibility by genomic neighbor typing.” Nature Microbiology.Abstract
Surveillance of circulating drug resistant bacteria is essential for healthcare providers to deliver effective empiric antibiotic therapy. However, the results of surveillance may not be available on a timescale that is optimal for guiding patient treatment. Here we present a method for inferring characteristics of an unknown bacterial sample by identifying the presence of sequence variation across the genome that is linked to a phenotype of interest, in this case drug resistance. We demonstrate an implementation of this principle using sequence k-mer content, matched to a database of known genomes. We show this technique can be applied to data from an Oxford Nanopore device in real time and is capable of identifying the presence of a known resistant strain in 5 minutes, even from a complex metagenomic sample. This flexible approach has wide application to pathogen surveillance and may be used to greatly accelerate diagnoses of resistant infections.
DR MacFadden, BCoburn, K Břinda, A Corbeil, N Daneman, D Fisman, RS Lee, M. Lipsitch, RG Melano, S Mubareka, and WP Hanage. In Press. “Using the Association Between Antibiotic Susceptibility and Genetic Relatedness to Rescue Old Drugs for Empiric Use.” Antimicrobial Agents and Chemotherapy.
In Preparation
B Cheng, MA Behr, D Grenier, BP Howden, T Cohen, and RS Lee. In Preparation. “Whole genome sequencing for epidemiological studies of tuberculosis: a systematic review of applications and reporting practices”.
Submitted
RS Lee et al. Submitted. “Genomic Epidemiology of Methicillin-Resistant Staphylococcus aureus in Two Cohorts of High-Risk Military Trainees.” medRvix.
NL Sherry, RS Lee, CL Gorrie, JC Kwong, RL Stuart, T Korman, C Marshall, C Higgs, HT Chan, M Graham, PDR Johnson, M Leroi, C Reed, M. Richards, MA Slavin, LJ Worth, BP Howden, and ML Grayson. Submitted. “Genomic interrogation of the burden and transmission of multidrug-resistant pathogens within and across hospital networks.” bioRvix.
G Alvarez, A Zwerling, C Duncan, C Pease, D Van Dyk, MA Behr, RS Lee, M Mulpuru, S Pakhale, DW Cameron, SD Aaron, M Patterson, J Allen, K Sullivan, A Jolly, M Sharma, and FB Jamieson. Submitted. “Molecular epidemiology of Mycobacterium tuberculosis to describe the transmission dynamics among Inuit in Iqaluit Nunavut using whole genome sequencing”.
2020
RS Lee, JF Proulx, F McIntosh, MA Behr, and WP Hanage. 2/4/2020. “Previously-undetected superspreading of Mycobacterium tuberculosis revealed by deep sequencing.” eLife.Abstract
Tuberculosis disproportionately affects the Canadian Inuit. To address this, it is imperative we understand transmission dynamics in this population. We investigate whether ‘deep’ sequencing can provide additional resolution compared to standard sequencing, using a well-characterized outbreak from the Arctic (2011-2012, 50 cases). Samples were sequenced to ~500-1000x and reads were aligned to a novel local reference genome generated with PacBio SMRT sequencing. Consensus and heterogeneous variants were identified and compared across genomes. In contrast with previous genomic analyses using ~50x depth, deep sequencing allowed us to identify a novel super-spreader who likely transmitted to up to 17 other cases during the outbreak (35% of all cases that year). It is increasingly evident that within-host diversity should be incorporated into transmission analyses; deep sequencing may facilitate more accurate detection of super-spreaders and transmission clusters. This has implications not only for TB, but all genomic studies of transmission - regardless of pathogen.
2019
Matthew P. Cheng, Robyn S. Lee, Alexandre P. Cheng, Samuel De L'Etoile-Morel, Koray Demir, Cedric P. Yansouni, Patrick Harris, Emily G. McDonald, and Todd C. Lee. 5/23/2019. “Beta-Lactam/Beta-Lactamase Inhibitor Therapy for Potential AmpC-Producing Organisms: A Systematic Review and Meta-Analysis.” Open Forum Infectious Diseases. Publisher's Version
Sarah Ackley, Robyn S Lee, Lee Worden, Erin Zwick, Travis C Porco, Marcel A Behr, and Caitlin Pepperell. 3/20/2019. “Multiple Exposures, Reinfection, and Risk of Progression to Active Tuberculosis.” Royal Society Open Science. Publisher's VersionAbstract
A recent study reported on a tuberculosis outbreak in a largely Inuit village. Among recently infected individuals, exposure to additional active cases was associated with an increasing probability of developing active disease within a year. Using binomial risk models, we evaluated two potential mechanisms by which multiple infections during the first year following initial infection could account for increasing disease risk with increasing exposures. In the reinfection model, multiple exposures have an independent risk of becoming an infection, and infections contribute independently to active disease. In the threshold model, disease risk follows a sigmoidal function with small numbers of exposures conferring a low risk of active disease and large numbers of exposures conferring a high risk. To determine the dynamic impact of reinfection during the early phase of infection, we performed simulations from a modified Reed-Frost model of TB dynamics following spread from an initial number of cases. We parameterized this model with the maximum likelihood estimates from the reinfection and threshold models in addition to the observed distribution of exposures among recent infections. We find that both models can plausibly account for the observed increase in disease risk with increasing exposures, but the threshold model confers a better fit than a nested model without a threshold (p=0.04). Our simulations indicate that multiple exposures during this critical time period can lead to dramatic increases in outbreak size. In order to decrease TB burden in high-prevalence settings, it may be necessary to implement measures aimed at preventing repeated exposures, in addition to preventing primary infection.
2018
Michael Martin, Robyn S Lee, Lauren A Cowley, Jennifer L Gardy, and William P Hanage. 10/11/2018. “Within-host diversity and its utility for Mycobacterium tuberculosis transmission inferences.” Microbial Genomics. Publisher's VersionAbstract

Whole genome sequencing in conjunction with traditional epidemiology has been used to reconstruct transmission networks of Mycobacterium tuberculosisduring outbreaks. Given its low mutation rate, genetic diversity within M. tuberculosis outbreaks can be extremely limited – making it difficult to determine precisely who transmitted to whom. In addition to consensus single nucleotide polymorphisms (cSNPs), examining heterogeneous alleles (hSNPs) has been proposed to improve resolution. However, few studies have examined the potential biases in detecting these hSNPs. Here, we analyzed genome sequence data from 25 specimens from British Columbia, Canada. Specimens were sequenced to a depth of 112-296x. We observed biases in the read depth, base quality, strand distribution, and read placement where possible hSNPs were initially identified, so we applied conservative filters to reduce false positives. Overall, there was phylogenetic concordance between the observed 2542 cSNP and 63 hSNP loci. Furthermore, we identified hSNPs shared exclusively by epidemiologically linked patients, supporting their use in transmission inferences. We conclude that hSNPs may add resolution to transmission networks, particularly where the overall genetic diversity is low.

Robyn S Lee, Anders Goncalves da Silva, Sarah L Baines, Susan Ballard, Glen P Carter, Janet Strachan, Jason C Kwong, Mark Schultz, Dieter Bulach, Timothy P Stinear, and Benjamin P Howden. 9/4/2018. “The changing landscape of vancomycin-resistant Enterococcus faecium in Australia: A population-level genomic study.” J Antimicrob Chemother. Publisher's VersionAbstract

Background.Vancomycin-resistant Enterococcus faecium(VREfm) represent a major source of nosocomial infection worldwide. In Australia, there has been a recent concerning increase in bacteremia associated with the vanAgenotype, prompting investigation into the genomic epidemiology of VREfm.

Methods. A population-level study of VREfm(Nov.10th- Dec.9th, 2015). 321 VREfm isolates (from 286 patients) across Victoria State were collected and sequenced with Illumina NextSeq. SNPs were used to assess relatedness. STs and genes associated with resistance and virulence were identified. The vanA-harbouring plasmid from an isolate from each ST was assembled using long-read data. Illumina reads from remaining isolates were then mapped to these assemblies to identify their probable vanA-harbouring plasmid.

Results.vanA-VREfm comprised 17.8% of isolates. ST203, ST80 and a pstS(-) clade, ST1421, predominated (30.5%, 30.5% and 37.2%, respectively). Most vanB-VREfm were ST796 (77.7%). vanA-VREfm were more closely-related within hospitals versus between them (core SNPs 10 [IQR 1-357] versus 356 [179-416] respectively), suggesting discrete introductions of vanA-VREfm, with subsequent intra-hospital transmission. In contrast, vanB-VREfm had similar core SNP distributions within versus between hospitals, due to widespread dissemination of ST796.Different vanA-harbouring plasmids were found across most STs. With exception of ST78 and ST796, Tn1546transposons also varied. Phylogenetic analysis revealed Australian strains were often interspersed with those from other countries, suggesting ongoing cross-continental transmission.

Conclusions.Emerging vanA-VREfm in Australia is polyclonal, indicating repeat introductions of vanA-VREfm into hospitals and subsequent dissemination. The close relationship to global strains reinforces the need for ongoing screening and control of VREfm in Australia and abroad.

Robyn S Lee and Benjamin P Howden. 2018. “The Critical Importance of Sampling Fraction to Inferences of Mycobacterium tuberculosis Transmission.” Clin Infect Dis, 66, 1, Pp. 159-160.
Robyn S Lee, Torsten Seemann, Helen Heffernan, Jason C Kwong, Anders Gonçalves da Silva, Glen P Carter, Rosemary Woodhouse, Kristin H Dyet, Dieter M Bulach, Timothy P Stinear, Benjamin P Howden, and Deborah A Williamson. 2018. “Genomic epidemiology and antimicrobial resistance of Neisseria gonorrhoeae in New Zealand.” J Antimicrob Chemother, 73, 2, Pp. 353-364.Abstract
Background: Antimicrobial-resistant Neisseria gonorrhoeae is a major threat to public health. No studies to date have examined the genomic epidemiology of gonorrhoea in the Western Pacific Region, where the incidence of gonorrhoea is particularly high. Methods: A population-level study of N. gonorrhoeae in New Zealand (October 2014 to May 2015). Comprehensive susceptibility testing and WGS data were obtained for 398 isolates. Relatedness was inferred using phylogenetic trees, and pairwise core SNPs. Mutations and genes known to be associated with resistance were identified, and correlated with phenotype. Results: Eleven clusters were identified. In six of these clusters, >25% of isolates were from females, while in eight of them, >15% of isolates were from females. Drug resistance was common; 98%, 32% and 68% of isolates were non-susceptible to penicillin, ciprofloxacin and tetracycline, respectively. Elevated MICs to extended-spectrum cephalosporins (ESCs) were observed in 3.5% of isolates (cefixime MICs ≥ 0.12 mg/L, ceftriaxone MICs ≥ 0.06 mg/L). Only nine isolates had penA XXXIV genotypes, three of which had decreased susceptibility to ESCs (MIC = 0.12 mg/L). Azithromycin non-susceptibility was identified in 43 isolates (10.8%); two of these isolates had 23S mutations (C2611T, 4/4 alleles), while all had mutations in mtrR or its promoter. Conclusions: The high proportion of females in clusters suggests transmission is not exclusively among MSM in New Zealand; re-assessment of risk factors for transmission may be warranted in this context. As elevated MICs of ESCs and/or azithromycin were found in closely related strains, targeted public health interventions to halt transmission are urgently needed.
2017
Robyn S Lee and Madhukar Pai. 2017. “Real-Time Sequencing of Mycobacterium tuberculosis: Are We There Yet?” J Clin Microbiol, 55, 5, Pp. 1249-1254.Abstract
Whole-genome sequencing has taken a leading role in epidemiologic studies of tuberculosis, but thus far, its real-time clinical utility has been low, in part because of the requirement for culture. In their report in this issue, Votintseva et al. (A. A. Votintseva, P. Bradley, L. Pankhurst, C. del Ojo Elias, M. Loose, K. Nilgiriwala, A. Chatterjee, E. G. Smith, N. Sanderson, T. M. Walker, M. R. Morgan, D. H. Wyllie, A. S. Walker, T. E. A. Peto, D. W. Crook, and Z. Iqbal, J Clin Microbiol 55:1285-1298, 2017, https://doi.org/10.1128/JCM.02483-16) present a new method for extracting DNA directly from smear-positive respiratory samples, making it feasible to generate drug resistance predictions and phylogenetic trees in 44 h with the Illumina MiSeq. They also illustrate the potential for a <24-h turnaround time from DNA extraction to clinically relevant results with Illumina MiniSeq and Oxford Nanopore Technologies MinION. We comment on the promise and limitations of these approaches.
2016
Robyn S Lee and Marcel A Behr. 2016. “Does Choice Matter? Reference-Based Alignment for Molecular Epidemiology of Tuberculosis.” J Clin Microbiol, 54, 7, Pp. 1891-1895.Abstract
When using genome sequencing for molecular epidemiology, short sequence reads are aligned to an arbitrary reference strain to detect single nucleotide polymorphisms. We investigated whether reference genome selection influences epidemiological inferences of Mycobacterium tuberculosis transmission by aligning sequence reads from 162 closely related lineage 4 (Euro-American) isolates to 7 different genomes. Phylogenetic trees were consistent with use of all but the most divergent genomes, suggesting that reference choice can be based on considerations other than M. tuberculosis lineage.
Faiz Ahmad Khan, Greg J Fox, Robyn S Lee, Mylene Riva, Andrea Benedetti, Jean-François Proulx, Shelley Jung, Karen Hornby, Marcel A Behr, and Dick Menzies. 2016. “Housing and tuberculosis in an Inuit village in northern Quebec: a case-control study.” CMAJ Open, 4, 3, Pp. E496-E506.Abstract
BACKGROUND: Between November 2011 and November 2012, an Inuit village in Nunavik, Quebec experienced a surge in the occurrence of active TB; contact investigations showed that TB infection was highly prevalent (62.6%), particularly among those over age 14 years (78.8%). A nested case-control study showed that nutritional inadequacy was associated with acquisition of infection but not progression to disease. We performed a study to determine whether characteristics of one's dwelling were associated with 1) acquisition of newly diagnosed TB infection and 2) progression to confirmed or probable disease among those with TB infection. METHODS: In this nested case-control study, we enrolled 200 people who were household or social contacts of at least 1 person with active TB or had received a diagnosis of active TB and assessed whether characteristics of their dwellings were associated with their odds of having newly diagnosed TB infection and/or odds of progression to disease between November 2011 and November 2012. For our first objective, we compared participants with newly diagnosed TB infection (regardless of their disease status) to a control group of contacts who were uninfected. For the second objective, we compared participants with confirmed or probable disease to a control group consisting of those with infection but no disease. We used information collected during investigation of the contacts and from study questionnaires to determine whether participants may have been exposed to TB in their own home (if they had shared a dwelling with someone who had smear-positive TB during the outbreak) or in other dwellings that they visited at least weekly. RESULTS: The participants lived in 79 dwellings. The mean number of people per room was 1.1 (standard deviation [SD] 0.5). The mean room size and ventilation level of the common living space (kitchen and living/dining rooms) were 67.9 (SD 9.4) m3 and 1.69 (SD 0.26) air changes per hour, respectively. After adjustment for potential confounders, the number of people per room was positively associated with the odds of newly diagnosed infection and odds of disease, but only among participants who lived with someone with smear-positive TB (the minority of participants). Other dwelling characteristics were not associated with either outcome. INTERPRETATION: Reducing household crowding may contribute to TB prevention. Overall, our investigations have not identified associations that explain the elevated disease risk in this village. In light of our results and considering the high prevalence of TB infection, treatment of latent infection is an essential intervention for long-term reduction of TB incidence in this village.
Robyn S Lee and Marcel A Behr. 2016. “The implications of whole-genome sequencing in the control of tuberculosis.” Ther Adv Infect Dis, 3, 2, Pp. 47-62.Abstract
The availability of whole-genome sequencing (WGS) as a tool for the diagnosis and clinical management of tuberculosis (TB) offers considerable promise in the fight against this stubborn epidemic. However, like other new technologies, the best application of WGS remains to be determined, for both conceptual and technical reasons. In this review, we consider the potential value of WGS in the clinical laboratory for the detection of Mycobacterium tuberculosis and the prediction of antibiotic resistance. We also discuss issues pertaining to data generation, interpretation and dissemination, given that WGS has to date been generally performed in research labs where results are not necessarily packaged in a clinician-friendly format. Although WGS is far more accessible now than it was in the past, the transition from a research tool to study TB into a clinical test to manage this disease may require further fine-tuning. Improvements will likely come through iterative efforts that involve both the laboratories ready to move TB into the genomic era and the front-line clinical/public health staff who will be interpreting the results to inform management decisions.
Robyn S Lee, Jean-François Proulx, Dick Menzies, and Marcel A Behr. 2016. “Progression to tuberculosis disease increases with multiple exposures.” Eur Respir J, 48, 6, Pp. 1682-1689.Abstract
During a single year, a Canadian village had 34 individuals with microbiologically confirmed tuberculosis (TB) among 169 people with a new infection (20%). A contact investigation revealed multiple exposures for each person. We investigated whether the intensity of exposure might contribute to this extraordinary risk of disease.We carried out a case-control study using a public health database. Among those with a new infection, 34 had culture-confirmed TB (cases) and 118 did not progress to disease (controls). 17 patients with probable disease were excluded. Contact investigation data were utilised to tabulate the number of potential sources (total exposures). Generalised estimating equations with a logit link were used to identify associations between exposures and progression, and to investigate other potential risk factors.The median (interquartile range) number of total exposures was 15 (3-23) for cases and 3 (2-12) for controls (p=0.001). The adjusted OR for disease was 1.11 (95% CI 1.06-1.16) per additional exposure, corresponding to an OR of 3.4 for disease when comparing the medians of 15 versus 3 total exposures. This association increased when restricting to tuberculin skin test conversions.Increased exposure could be a marker of greater risk of progression to TB disease. Therefore, this risk may not be transportable across epidemiologic settings with variable exposure intensities.
2015
Gregory J Fox, Robyn S Lee, Michel Lucas, Faiz Ahmad Khan, Jean-Francois Proulx, Karen Hornby, Shelley Jung, Andrea Benedetti, Marcel A Behr, and Dick Menzies. 2015. “Inadequate Diet Is Associated with Acquiring Mycobacterium tuberculosis Infection in an Inuit Community. A Case-Control Study.” Ann Am Thorac Soc, 12, 8, Pp. 1153-62.Abstract
BACKGROUND: Tuberculosis predominantly affects socioeconomically disadvantaged communities. The extent to which specific dietary and lifestyle factors contribute to tuberculosis susceptibility has not been established. METHODS: A total of 200 residents of a village in Northern Quebec were investigated during a tuberculosis outbreak and identified to have active tuberculosis, latent tuberculosis infection, or neither. Participants completed questionnaires about their intake of food from traditional and commercial sources, and provided blood samples. Adults were asked about recent smoking and drug and alcohol intake. Nutritional adequacy was evaluated with reference to North American standards. Multiple dietary, lifestyle, and housing factors were combined in a logistic regression model evaluating the contributions of each to disease and infection. FINDINGS: After adjusting for potential confounding, new infection was associated with inadequate intake of fruit and vegetables (odds ratio [OR], 2.1; 95% confidence interval [CI], 1.03-4.3), carbohydrates (OR, 4.4; 95% CI, 1.2-16.3), and certain vitamins and minerals. A multivariable model, combining nutrition, housing, and lifestyle factors, found associations between new infection and inadequate fruit and vegetable intake (OR, 2.3; 95% CI, 1.0-5.1), living in the same house as a person with smear-positive tuberculosis (OR, 14.7; 95% CI, 1.6-137.3), and visiting a community gathering house (OR, 3.7; 95% CI, 1.7-8.3). Current smoking was associated with new infection (OR, 9.4; 95% CI, 1.2-72) among adults completing a detailed lifestyle survey. INTERPRETATION: Inadequate nutrition was associated with increased susceptibility to infection, but not active tuberculosis. Interventions addressed at improving nutrition may reduce susceptibility to infection in settings where access to healthy foods is limited.

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