Biology subjects

Grandjean, L.

Publications and source records attributed to Grandjean, L..

7 recordsLinked to original sources

bioRxiv2024

Tiled Amplicon Sequencing Enables Culture-free Whole-Genome Sequencing of Pathogenic Bacteria From Clinical Specimens

Pathogen sequencing is an important tool for disease surveillance and demonstrated its high value during the COVID-19 pandemic. Viral sequencing during the pandemic allowed us to track disease spread, quickly identify new variants, and guide the development of vaccines. Tiled amplicon sequencing, in which a panel of primers is used for multiplex amplification of fragments across an entire genome, was the cornerstone of SARS-CoV-2 sequencing. The speed, reliability, and cost-effectiveness of this method led to its implementation in academic and public health laboratories across the world and adaptation to a broad range of viral pathogens. However, similar methods are not available for larger bacterial genomes, for which whole-genome sequencing typically requires in vitro culture. This increases costs, error rates and turnaround times. The need to culture poses particular problems for medically important bacteria such as Mycobacterium tuberculosis, which are slow to grow and challenging to culture. As a proof of concept, we developed two novel whole-genome amplicon panels for M. tuberculosis and Streptococcus pneumoniae. Applying our amplicon panels to clinical samples, we show the ability to classify pathogen subgroups and to reliably identify markers of drug resistance without culturing. Development of this work in clinical settings has the potential to dramatically reduce the time of diagnosis of drug resistance for multiple drugs in parallel, enabling earlier intervention for high priority pathogens.

genomics↗

bioRxiv2024

A new method for detecting mixed Mycobacterium tuberculosis infection and reconstructing constituent strains provides insights into transmission

BackgroundMixed infection with multiple strains of the same pathogen in a single host can present clinical and analytical challenges. Whole genome sequence (WGS) data can identify signals of multiple strains in samples, though the precision of previous methods can be improved. Here, we present MixInfect2, a new tool to accurately detect mixed samples from Mycobacterium tuberculosis WGS data. We then evaluate three approaches for reconstructing the underlying mixed constituent strain sequences. This allows these samples to be included in downstream analysis to gain insights into the epidemiology and transmission of mixed infections. MethodsWe employed a Gaussian mixture model to cluster allele frequencies at mixed sites (hSNPs) in each sample to identify signals of multiple strains. Building upon our previous tool, MixInfect, we increased the accuracy of classifying in vitro mixed samples through multiple improvements to the bioinformatic pipeline. Major and minor proportion constituent strains were reconstructed using three approaches and assessed by comparing the estimated sequence to the known constituent strain sequence. Lastly, mixed infections in a real-world Mycobacterium tuberculosis population from Moldova were detected with MixInfect2 and clusters of recent transmission that included major and minor constituent strains were built. ResultsAll 36/36 in vitro mixed and 12/12 non-mixed samples were correctly classified with MixInfect2, and major strain proportions estimated with high accuracy, outperforming previous tools. Reconstructed major strain sequences closely matched the true constituent sequence by taking the allele at the highest frequency at hSNPs, while the best performing approach to reconstruct the minor proportion strain sequence was identifying the closest non-mixed isolate in the same population, though no approach was effective when the minor strain proportion was at 5%. Finally, fewer mixed infections were identified in Moldova than previous estimates (6.6% vs 17.4%) and we found multiple instances where the constituent strains of mixed samples were present in transmission clusters. ConclusionsMixInfect2 accurately detects samples with evidence of mixed infection from WGS data and provides an excellent estimate of the mixture proportions. While there are limitations in reconstructing the constituent strain sequences of mixed samples, we present recommendations for the best approach to include these isolates in further analyses.

genomics↗

bioRxiv2022

Within-host diversity improves phylogenetic and transmission reconstruction of SARS-CoV-2 outbreaks

Accurate inference of who infected whom in an infectious disease outbreak is critical for the delivery of effective infection prevention and control. The increased resolution of pathogen whole-genome sequencing has significantly improved our ability to infer transmission events. Despite this, transmission inference often remains limited by the lack of genomic variation between the source case and infected contacts. Although within-host genetic diversity is common among a wide variety of pathogens, conventional whole-genome sequencing phylogenetic approaches to reconstruct outbreaks exclusively use consensus sequences, which consider only the most prevalent nucleotide at each position and therefore fail to capture low frequency variation within samples. We hypothesized that including within-sample variation in a phylogenetic model would help to identify who infected whom in instances in which this was previously impossible. Using whole-genome sequences from SARS-CoV-2 multi-institutional outbreaks as an example, we show how within-sample diversity is stable among repeated serial samples from the same host, is transmitted between those cases with known epidemiological links, and how this improves phylogenetic inference and our understanding of who infected whom. Our technique is applicable to other infectious diseases and has immediate clinical utility in infection prevention and control.

evolutionary biology↗

bioRxiv2020

Detection of a bedaquiline / clofazimine resistance reservoir in Mycobacterium tuberculosis predating the antibiotic era

Drug resistance in tuberculosis (TB) poses a major ongoing challenge to public health. The recent inclusion of bedaquiline into TB drug regimens has improved treatment outcomes, but this advance is threatened by the emergence of strains of Mycobacterium tuberculosis (Mtb) resistant to bedaquiline. Clinical bedaquiline resistance is most frequently conferred by off-target resistance-associated variants (RAVs) in the mmpR5 gene (Rv0678), the regulator of an efflux pump, which can also confer cross-resistance to clofazimine, another TB drug. We compiled a dataset of 3,682 Mtb genomes, including 150 carrying variants in mmpR5 that have been associated to borderline (henceforth intermediate) or confirmed resistance to bedaquiline. We identified eight cases where RAVs were present in the genomes of strains collected prior to the use of bedaquiline in TB treatment regimes. Phylogenetic reconstruction points to multiple emergence events and circulation of RAVs in mmpR5, some estimated to predate the introduction of bedaquiline. However, epistatic interactions can complicate bedaquiline drug-susceptibility prediction from genetic sequence data. Indeed, in one clade of isolates where the RAV Ile67fs is estimated to have emerged prior to the antibiotic era, co-occurrence of mutations in mmpL5 are found to neutralise bedaquiline resistance. The presence of a pre-existing reservoir of Mtb strains carrying bedaquiline RAVs prior to its clinical use augments the need for rapid drug susceptibility testing and individualised regimen selection to safeguard the use of bedaquiline in TB care and control.

genomics↗

bioRxiv2020

Evaluation of a novel multiplexed assay for determining IgG levels and functional activity to SARS-CoV-2.

BackgroundThe emergence of SARS-CoV-2 has led to the development of new serological assays that could aid in diagnosis and evaluation of seroprevalence to inform an understanding of the burden of COVID-19 disease. Many available tests lack rigorous evaluation and therefore results may be misleading. ObjectivesThe aim of this study was to assess the performance of a novel multiplexed immunoassay for the simultaneous detection of antibodies against SARS-CoV-2 trimeric spike (S), spike receptor binding domain (RBD), spike N terminal domain and nucleocapsid antigen and a novel pseudo-neutralisation assay. MethodsA multiplexed solid-phase chemiluminescence assay (Meso Scale Discovery) was evaluated for the simultaneous detection of IgG binding to four SARS-CoV-2 antigens and the quantification of antibody-induced ACE-2 binding inhibition (pseudo-neutralisation assay). Sensitivity was evaluated with a total of 196 COVID-19 serum samples (169 confirmed PCR positive and 27 anti-nucleocapsid IgG positive) from individuals with mild symptomatic or asymptomatic disease. Specificity was evaluated with 194 control serum samples collected from adults prior to December 2019. ResultsThe specificity and sensitivity of the binding IgG assay was highest for S protein with a specificity of 97.4% and sensitivity of 96.2% for samples taken 14 days and 97.9% for samples taken 21 days following the onset of symptoms. IgG concentration to S and RBD correlated strongly with percentage inhibition measured by the pseudo-neutralisation assay. ConclusionExcellent sensitivity for IgG detection was obtained over 14 days since onset of symptoms for three SARS-CoV-2 antigens (S, RBD and N) in this multiplexed assay which can also measure antibody functionality.

immunology↗

bioRxiv2018

Isoniazid preventive therapy protects against tuberculosis among household contacts of isoniazid-resistant patients.

BackgroundThe World Health Organization recommends the use of isoniazid (INH) alone or combination INH and rifapentine therapy to treat latent tuberculosis infection (LTBI) in groups at high risk of tuberculosis (TB) progression. The recent rise of INH- and multi-drug resistant (MDR) TB has complicated the choice of LTBI treatment regimen. We examine the risk of TB disease among household contacts (HHCs) who received INH after being exposed to patients with drug-sensitive, INH-resistant, or MDR tuberculosis. MethodsIn this prospective cohort study conducted Between September 2009 and August 2012 in Lima, Peru, we identified 4,500 index TB patients and measured incident TB disease in their 14,044 HHCs over a one-year follow-up period. HHCs under 19 years of age were offered INH preventive therapy (IPT). We used a Cox frailty proportional hazards model to evaluate whether the effect of IPT on incident TB disease varied by the resistance profile of the index case. We repeated the analyses in a second independent dataset. FindingsAmong 4,216 HHCs under 19 years of age, 2,106 (50%) initiated IPT at enrolment. The protective effect of INH was more extreme in HHCs exposed to drug-sensitive (Hazard Ratio [95% confidence interval]=0{middle dot}2[0{middle dot}20-0{middle dot}50]) and to MDR-TB (0{middle dot}26[0{middle dot}08-0{middle dot}77]) compared to those exposed to mono-INH-resistant (0{middle dot}80[0{middle dot}23 to 2{middle dot}79]). Among those who received at least three months of INH, effectiveness increased across all three groups (INH-sensitive:0{middle dot}20 [0{middle dot}10 to 040]; MDR:0{middle dot}16 [0 02-127]; mono-INH-resistant:0{middle dot}72 [0{middle dot}16-3{middle dot}16]). In the second independent study, TB occurred in none of the 76 HHCs who received IPT compared to 3% (8/273) of those who did not. InterpretationWe found that IPT use is associated with reduced incidence of TB disease among contacts of MDR-TB patients. This finding suggests that INH may have role in the management of MDR-LTBI. FundingNational Institutes of Health and the National Institute of Allergy and Infectious Diseases CETR (U19AI109755) and TBRU (U19AI111224)

epidemiology↗

bioRxiv2017

The relative fitness of drug resistant Mycobacterium tuberculosis: a modelling study of household transmission in Lima, Peru

The relative fitness of drug resistant versus susceptible bacteria in an environment dictates resistance prevalence. Estimates for the relative fitness of resistant Mycobacterium tuberculosis (Mtb) strains are highly heterogeneous and mostly derived from in-vitro experiments. Measuring fitness in the field allows us to determine how the environment influences resistance spread.\n\nWe designed a household structured, stochastic mathematical model to estimate the fitness costs associated with multi-drug resistance (MDR) carriage in Mtb in Lima, Peru between 2010-2013. By fitting the model to data from a large prospective cohort study of TB disease in household contacts we estimated the fitness, relative to susceptible strains with a fitness of 1, of MDR-Mtb to be 0.33 (95% credible interval: 0.17-0.54) or 0.39 (0.26-0.58), if only transmission or progression to disease, respectively, was affected. The relative fitness of MDR-Mtb increased to 0.57 (0.43-0.73) when the fitness cost influenced both transmission and progression to disease equally.\n\nWe found the average relative fitness of MDR-Mtb circulating within households in Lima, Peru between 2010-2013 to be significantly lower than concurrent susceptible-Mtb. If these fitness levels do not change, then existing TB control programmes are likely to keep MDR-TB prevalence at current levels in Lima, Peru.

epidemiology↗