Within-host Diversity Research Articles

Dynamics of Human Immunodeficiency Virus-1 Genetic Diversification During Acute Infection

We analyzed human immunodeficiency virus envelope diversity in 98 acute infections. The within-host genetic diversity, divergence from transmitted/founder (T/F) strain, and the observed frequency of multiple T/F infections increased with Fiebig stage. These data identify rapid viral dynamics during acute infection with implications for clinical trials conducted in this setting.

Evaluating the Performance of Malaria Genetics for Inferring Changes in Transmission Intensity Using Transmission Modeling.

Substantial progress has been made globally to control malaria, however there is a growing need for innovative new tools to ensure continued progress. One approach is to harness genetic sequencing and accompanying methodological approaches as have been used in the control of other infectious diseases. However, to utilize these methodologies for malaria, we first need to extend the methods to capture the complex interactions between parasites, human and vector hosts, and environment, which all impact the level of genetic diversity and relatedness of malaria parasites. We develop an individual-based transmission model to simulate malaria parasite genetics parameterized using estimated relationships between complexity of infection and age from five regions in Uganda and Kenya. We predict that cotransmission and superinfection contribute equally to within-host parasite genetic diversity at 11.5% PCR prevalence, above which superinfections dominate. Finally, we characterize the predictive power of six metrics of parasite genetics for detecting changes in transmission intensity, before grouping them in an ensemble statistical model. The model predicted malaria prevalence with a mean absolute error of 0.055. Different assumptions about the availability of sample metadata were considered, with the most accurate predictions of malaria prevalence made when the clinical status and age of sampled individuals is known. Parasite genetics may provide a novel surveillance tool for estimating the prevalence of malaria in areas in which prevalence surveys are not feasible. However, the findings presented here reinforce the need for patient metadata to be recorded and made available within all future attempts to use parasite genetics for surveillance.

Inferring transmission heterogeneity using virus genealogies: Estimation and targeted prevention.

Spread of HIV typically involves uneven transmission patterns where some individuals spread to a large number of individuals while others to only a few or none. Such transmission heterogeneity can impact how fast and how much an epidemic spreads. Further, more efficient interventions may be achieved by taking such transmission heterogeneity into account. To address these issues, we developed two phylogenetic methods based on virus sequence data: 1) to generally detect if significant transmission heterogeneity is present, and 2) to pinpoint where in a phylogeny high-level spread is occurring. We derive inference procedures to estimate model parameters, including the amount of transmission heterogeneity, in a sampled epidemic. We show that it is possible to detect transmission heterogeneity under a wide range of simulated situations, including incomplete sampling, varying levels of heterogeneity, and including within-host genetic diversity. When evaluating real HIV-1 data from different epidemic scenarios, we found a lower level of transmission heterogeneity in slowly spreading situations and a higher level of heterogeneity in data that included a rapid outbreak, while R0 and Sackin's index (overall tree shape statistic) were similar in the two scenarios, suggesting that our new method is able to detect transmission heterogeneity in real data. We then show by simulations that targeted prevention, where we pinpoint high-level spread using a coalescence measurement, is efficient when sequence data are collected in an ongoing surveillance system. Such phylogeny-guided prevention is efficient under both single-step contact tracing as well as iterative contact tracing as compared to random intervention.

Within-host microevolution of Streptococcus pneumoniae is rapid and adaptive during natural colonisation

Genomic evolution, transmission and pathogenesis of Streptococcus pneumoniae, an opportunistic human-adapted pathogen, is driven principally by nasopharyngeal carriage. However, little is known about genomic changes during natural colonisation. Here, we use whole-genome sequencing to investigate within-host microevolution of naturally carried pneumococci in ninety-eight infants intensively sampled sequentially from birth until twelve months in a high-carriage African setting. We show that neutral evolution and nucleotide substitution rates up to forty-fold faster than observed over longer timescales in S. pneumoniae and other bacteria drives high within-host pneumococcal genetic diversity. Highly divergent co-existing strain variants emerge during colonisation episodes through real-time intra-host homologous recombination while the rest are co-transmitted or acquired independently during multiple colonisation episodes. Genic and intergenic parallel evolution occur particularly in antibiotic resistance, immune evasion and epithelial adhesion genes. Our findings suggest that within-host microevolution is rapid and adaptive during natural colonisation.

Sampling and summarizing transmission trees with multi-strain infections.

MotivationThe combination of genomic and epidemiological data holds the potential to enable accurate pathogen transmission history inference. However, the inference of outbreak transmission histories remains challenging due to various factors such as within-host pathogen diversity and multi-strain infections. Current computational methods ignore within-host diversity and/or multi-strain infections, often failing to accurately infer the transmission history. Thus, there is a need for efficient computational methods for transmission tree inference that accommodate the complexities of real data.ResultsWe formulate the direct transmission inference (DTI) problem for inferring transmission trees that support multi-strain infections given a timed phylogeny and additional epidemiological data. We establish hardness for the decision and counting version of the DTI problem. We introduce Transmission Tree Uniform Sampler (TiTUS), a method that uses SATISFIABILITY to almost uniformly sample from the space of transmission trees. We introduce criteria that prioritize parsimonious transmission trees that we subsequently summarize using a novel consensus tree approach. We demonstrate TiTUS’s ability to accurately reconstruct transmission trees on simulated data as well as a documented HIV transmission chain.Availability and implementation https://github.com/elkebir-group/TiTUS.Supplementary information Supplementary data are available at Bioinformatics online.

Within-Host Viral Diversity: A Window into Viral Evolution.

The evolutionary dynamics of a virus can differ within hosts and across populations. Studies of within-host evolution provide an important link between experimental studies of virus evolution and large-scale phylodynamic analyses. They can determine the extent to which global processes are recapitulated on local scales and how accurately experimental infections model natural ones. They may also inform epidemiologic models of disease spread and reveal how host-level dynamics contribute to a virus's evolution at a larger scale. Over the last decade, advances in viral sequencing have enabled detailed studies of viral genetic diversity within hosts. I review how within-host diversity is sampled, measured, and expressed, and how comparative studies of viral diversity can be leveraged to elucidate a virus's evolutionary dynamics. These concepts are illustrated with detailed reviews of recent research on the within-host evolution of influenza virus, dengue virus, and cytomegalovirus.

A de novo approach to inferring within-host fitness effects during untreated HIV-1 infection.

In the absence of effective antiviral therapy, HIV-1 evolves in response to the within-host environment, of which the immune system is an important aspect. During the earliest stages of infection, this process of evolution is very rapid, driven by a small number of CTL escape mutations. As the infection progresses, immune escape variants evolve under reduced magnitudes of selection, while competition between an increasing number of polymorphic alleles (i.e., clonal interference) makes it difficult to quantify the magnitude of selection acting upon specific variant alleles. To tackle this complex problem, we developed a novel multi-locus inference method to evaluate the role of selection during the chronic stage of within-host infection. We applied this method to targeted sequence data from the p24 and gp41 regions of HIV-1 collected from 34 patients with long-term untreated HIV-1 infection. We identify a broad distribution of beneficial fitness effects during infection, with a small number of variants evolving under strong selection and very many variants evolving under weaker selection. The uniquely large number of infections analysed granted a previously unparalleled statistical power to identify loci at which selection could be inferred to act with statistical confidence. Our model makes no prior assumptions about the nature of alleles under selection, such that any synonymous or non-synonymous variant may be inferred to evolve under selection. However, the majority of variants inferred with confidence to be under selection were non-synonymous in nature, and in most cases were have previously been associated with either CTL escape in p24 or neutralising antibody escape in gp41. We also identified a putative new CTL escape site (residue 286 in gag), and a region of gp41 (including residues 644, 648, 655 in env) likely to be associated with immune escape. Sites inferred to be under selection in multiple hosts have high within-host and between-host diversity although not all sites with high between-host diversity were inferred to be under selection at the within-host level. Our identification of selection at sites associated with resistance to broadly neutralising antibodies (bNAbs) highlights the need to fully understand the role of selection in untreated individuals when designing bNAb based therapies.

Dynamics of within-host Mycobacterium tuberculosis diversity and heteroresistance during treatment

BackgroundStudying within-host genetic diversity of Mycobacterium tuberculosis (Mtb) in patients during treatment may identify adaptations to antibiotic and immune pressure. Understanding the significance of genetic heteroresistance, and more specifically heterozygous resistance-associated variants (RAVs), is clinically important given increasing use of rapid molecular tests and whole genome sequencing (WGS). MethodsWe analyse data from six studies in KwaZulu-Natal, South Africa. Most patients (>75%) had baseline rifampicin resistance. Sputum was collected for culture at baseline and at between two and nine intervals until month six. Positive cultures underwent WGS. Mixed infections and reinfections were excluded from analysis. FindingsBaseline Mtb overall genetic diversity (at treatment initiation or major change to regimen) was associated with cavitary disease, not taking antiretroviral therapy if HIV infected, infection with lineage 2 strains and absence of second-line drug resistance on univariate analyses. Baseline genetic diversity was not associated with six-month outcome. Genetic diversity increased from baseline to weeks one and two before returning to previous levels. Baseline genetic heteroresistance was most common for bedaquiline (6/10 [60%] of isolates with RAVs) and fluoroquinolones (9/62 [13%]). Most patients with heterozygous RAVs on WGS with sequential isolates available demonstrated RAV persistence or fixation (17/20, 85%). New RAVs emerged in 9/286 (3%) patients during treatment. We could detect low-frequency RAVs preceding emergent resistance in only one case, although validation of deep sequencing to detect rare variants is required. InterpretationIn this study of single-strain Mtb infections, baseline within-host bacterial genetic diversity did not predict outcome but may reveal adaptations to host and drug pressures. Predicting emergent resistance from low-frequency RAVs requires further work to separate transient from consequential mutations. FundingWellcome Trust, NIH/NIAID

A Turkey-origin H9N2 Avian Influenza Virus Shows Low Pathogenicity but Different Within-Host Diversity in Experimentally Infected Turkeys, Quail and Ducks.

Avian influenza virus (AIV) is a highly diverse and widespread poultry pathogen. Its evolution and adaptation may be affected by multiple host and ecological factors, which are still poorly understood. In the present study, a turkey-origin H9N2 AIV was used as a model to investigate the within-host diversity of the virus in turkeys, quail and ducks in conjunction with the clinical course, shedding and seroconversion. Ten birds were inoculated oculonasally with a dose of 106 EID50 of the virus and monitored for 14 days. Virus shedding, transmission and seroconversion were evaluated, and swabs collected at selected time-points were characterized in deep sequencing to assess virus diversity. In general, the virus showed low pathogenicity for the examined bird species, but differences in shedding patterns, seroconversion and clinical outcome were noted. The highest heterogeneity of the virus population as measured by the number of single nucleotide polymorphisms and Shannon entropy was found in oropharyngeal swabs from quail, followed by turkeys and ducks. This suggests a strong bottleneck was imposed on the virus during replication in ducks, which can be explained by its poor adaptation and stronger selection pressure in waterfowl. The high within-host virus diversity in quail with high level of respiratory shedding and asymptomatic course of infection may contribute to our understanding of the role of quail as an intermediate host for adaptation of AIV to other species of poultry. In contrast, low virus complexity was observed in cloacal swabs, mainly from turkeys, showing that the within-host diversity may vary between different replication sites. Consequences of these observations on the virus evolution and adaptation require further investigation.

Influenza B Viruses Exhibit Lower Within-Host Diversity than Influenza A Viruses in Human Hosts.

Influenza B virus (IBV) undergoes seasonal antigenic drift more slowly than influenza A virus, but the reasons for this difference are unclear. While the evolutionary dynamics of influenza viruses play out globally, they are fundamentally driven by mutation, reassortment, drift, and selection at the level of individual hosts. These processes have recently been described for influenza A virus, but little is known about the evolutionary dynamics of IBV during individual infections and transmission events. Here, we define the within-host evolutionary dynamics of IBV by sequencing virus populations from naturally infected individuals enrolled in a prospective, community-based cohort over 8,176 person-seasons of observation. Through analysis of high depth-of-coverage sequencing data from samples from 91 individuals with influenza B, we find that IBV accumulates lower genetic diversity than previously observed for influenza A virus during acute infections. Consistent with studies of influenza A viruses, the within-host evolution of IBVs is characterized by purifying selection and the general absence of widespread positive selection of within-host variants. Analysis of shared genetic diversity across 15 sequence-validated transmission pairs suggests that IBV experiences a tight transmission bottleneck similar to that of influenza A virus. These patterns of local-scale evolution are consistent with the lower global evolutionary rate of IBV.IMPORTANCE The evolution of influenza virus is a significant public health problem and necessitates the annual evaluation of influenza vaccine formulation to keep pace with viral escape from herd immunity. Influenza B virus is a serious health concern for children, in particular, yet remains understudied compared to influenza A virus. Influenza B virus evolves more slowly than influenza A virus, but the factors underlying this are not completely understood. We studied how the within-host diversity of influenza B virus relates to its global evolution by sequencing viruses from a community-based cohort. We found that influenza B virus populations have lower within-host genetic diversity than influenza A virus and experience a tight genetic bottleneck during transmission. Our work provides insights into the varying dynamics of influenza viruses in human infection.

Previously undetected super-spreading of Mycobacterium tuberculosis revealed by deep sequencing.

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 the remaining 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.

A snapshot of diversity: Intraclonal variation of Escherichia coli clones as commensals and pathogens

Whole-genome sequencing has enabled detailed studies on bacterial evolution during infection, but there is limited knowledge on intraclonal variation. In this study, we sought to provide a snapshot of the intraclonal diversity of Escherichia coli as both commensal in the faecal environment and pathogen during urinary tract infection, respectively. This was performed by whole-genome sequencing and analyses of single nucleotide polymorphisms (SNPs) and gene-content variation in ten isolates belonging to the same clone and isolated from rectal swabs or urine samples. We identified only one clone in eight of the nine urines sampled (89 %). In both the commensal and pathogenic state, the within-host diversity was limited with intraclonal SNP diversity of 0–2 non-synonymous SNPs for each clone. The genetic diversity showed variation in gene content in a range of 2–15 genes in total for all clones, including genes positioned on plasmids, and in the K- and O-antigen cluster. The observed SNP- and gene variation shows that sampling of one colony would be enough for surveillance, outbreak investigations and clonal evolution. However, for studies of adaptation during or between colonization and infection, this variation is relevant to consider.

Quantifying within-host diversity of H5N1 influenza viruses in humans and poultry in Cambodia.

Avian influenza viruses (AIVs) periodically cross species barriers and infect humans. The likelihood that an AIV will evolve mammalian transmissibility depends on acquiring and selecting mutations during spillover, but data from natural infection is limited. We analyze deep sequencing data from infected humans and domestic ducks in Cambodia to examine how H5N1 viruses evolve during spillover. Overall, viral populations in both species are predominated by low-frequency (<10%) variation shaped by purifying selection and genetic drift, and half of the variants detected within-host are never detected on the H5N1 virus phylogeny. However, we do detect a subset of mutations linked to human receptor binding and replication (PB2 E627K, HA A150V, and HA Q238L) that arose in multiple, independent humans. PB2 E627K and HA A150V were also enriched along phylogenetic branches leading to human infections, suggesting that they are likely human-adaptive. Our data show that H5N1 viruses generate putative human-adapting mutations during natural spillover infection, many of which are detected at >5% frequency within-host. However, short infection times, genetic drift, and purifying selection likely restrict their ability to evolve extensively during a single infection. Applying evolutionary methods to sequence data, we reveal a detailed view of H5N1 virus adaptive potential, and develop a foundation for studying host-adaptation in other zoonotic viruses.

Linking influenza virus evolution within and between human hosts.

Influenza viruses rapidly diversify within individual human infections. Several recent studies have deep-sequenced clinical influenza infections to identify viral variation within hosts, but it remains unclear how within-host mutations fare at the between-host scale. Here, we compare the genetic variation of H3N2 influenza within and between hosts to link viral evolutionary dynamics across scales. Synonymous sites evolve at similar rates at both scales, indicating that global evolution at these putatively neutral sites results from the accumulation of within-host variation. However, nonsynonymous mutations are depleted between hosts compared to within hosts, suggesting that selection purges many of the protein-altering changes that arise within hosts. The exception is at antigenic sites, where selection detectably favors nonsynonymous mutations at the global scale, but not within hosts. These results suggest that selection against deleterious mutations and selection for antigenic change are the main forces that act on within-host variants of influenza virus as they transmit and circulate between hosts.

Two-year longitudinal survey reveals high genetic diversity of Schistosoma mansoni with adult worms surviving praziquantel treatment at the start of mass drug administration in Uganda

BackgroundA key component of schistosomiasis control is mass drug administration with praziquantel. While control interventions have been successful in several endemic regions, mass drug administration has been less effective in others. Here we focus on the impact of repeated praziquantel treatment on the population structure and genetic diversity of Schistosoma mansoni.MethodsWe examined S. mansoni epidemiology, population genetics, and variation in praziquantel susceptibility in parasites isolated from children across three primary schools in a high endemicity region at the onset of the Ugandan National Control Programme. Children were sampled at 11 timepoints over two years, including one week and four weeks post-praziquantel treatment to evaluate short-term impacts on clearance and evidence of natural variation in susceptibility to praziquantel.ResultsPrevalence of S. mansoni was 85% at baseline. A total of 3576 miracidia larval parasites, isolated from 203 individual children, were genotyped at seven loci. Overall, genetic diversity was high and there was low genetic differentiation, indicating high rates of parasite gene flow. Schistosome siblings were found both pre-treatment and four weeks post-treatment, demonstrating adult worms surviving treatment and natural praziquantel susceptibility variation in these populations at the beginning of mass drug administration. However, we did not find evidence for selection on these parasites. While genetic diversity decreased in the short-term (four weeks post-treatment), diversity did not decrease over the entire period despite four rounds of mass treatment. Furthermore, within-host genetic diversity was affected by host age, host sex, infection intensity and recent praziquantel treatment.ConclusionsOur findings suggest that praziquantel treatments have short-term impacts on these parasite populations but impacts were transient and no long-term reduction in genetic diversity was observed. High gene flow reduces the likelihood of local adaptation, so even though parasites surviving treatment were observed, these were likely to be diluted at the beginning of the Ugandan National Control Programme. Together, these results suggest that MDA in isolation may be insufficient to reduce schistosome populations in regions with high genetic diversity and gene flow.

Deconvoluting the diversity of within-host pathogen strains in a multi-locus sequence typing framework

BackgroundBacterial pathogens exhibit an impressive amount of genomic diversity. This diversity can be informative of evolutionary adaptations, host-pathogen interactions, and disease transmission patterns. However, capturing this diversity directly from biological samples is challenging.ResultsWe introduce a framework for understanding the within-host diversity of a pathogen using multi-locus sequence types (MLST) from whole-genome sequencing (WGS) data. Our approach consists of two stages. First we process each sample individually by assigning it, for each locus in the MLST scheme, a set of alleles and a proportion for each allele. Next, we associate to each sample a set of strain types using the alleles and the strain proportions obtained in the first step. We achieve this by using the smallest possible number of previously unobserved strains across all samples, while using those unobserved strains which are as close to the observed ones as possible, at the same time respecting the allele proportions as closely as possible. We solve both problems using mixed integer linear programming (MILP). Our method performs accurately on simulated data and generates results on a real data set of Borrelia burgdorferi genomes suggesting a high level of diversity for this pathogen.ConclusionsOur approach can apply to any bacterial pathogen with an MLST scheme, even though we developed it with Borrelia burgdorferi, the etiological agent of Lyme disease, in mind. Our work paves the way for robust strain typing in the presence of within-host heterogeneity, overcoming an essential challenge currently not addressed by any existing methodology for pathogen genomics.

552. Within-Host Evaluation of Colonization During Active Methicillin-Resistant S. aureus Bacteremia

Background Staphylococcus aureus is a common commensal pathogen and a frequent cause of bacteremia, with nasal and blood isolates from single patients matching >80% of the time. Based on our prior work on paired single-patient isolates,1 our aim was to collect a contemporary set of colonizing isolates from those with methicillin-resistant S. aureus (MRSA) bloodstream infections (BSIs), evaluate the within-host diversity by whole-genome sequencing (WGS), and detail the clinical features linked to colonization.MethodsAdult patients with MRSA BSIs were screened for MRSA in the anterior nares from July 2018 to March 2019. Blood isolates underwent WGS, and spa and agr function screens were performed on three unique nasal isolates per patient. Clinical data from the electronic medical records underwent uni- and multivariate analyses on clinical features and outcomes.ResultsOf 55 unique patients with MRSA BSIs, screening of 45 subjects revealed that 67% were colonized with MRSA. The majority (64%) were males, 32% had prior colonization, and the most common infection sources were vascular access (27%), skin (24%), and unknown (24%). For those with nasal colonization, blood isolates were composed of 57% clonal complex (CC)5/t002, 33% CC8/t008, and 10% other. 81% of nasal isolates matched the blood, with 20% of nasal isolates harboring diverse spa types and 23% carried agr mutants. During this time frame, WGS found one transmission event involving a colonized subject. Colonization was associated with male gender (OR=4.52 95% CI [1.05–19.49]; P = 0.04) and prior hospital admission within the last 3 months (OR=6.12 95% CI [1.44–26.09]; P = 0.01) in multivariate analysis, with no differences in outcomes.ConclusionColonization is an important component of invasive MRSA disease, and we found high rates of colonization with a predominance of the CC5. We also noted significant diversity and high proportion of agr mutants. At-risk groups included males and those with prior hospitalization. Combined molecular and clinical analyses can define the intrahost and interhost transmission dynamics of MRSA, and enables the development of targeted approaches in order to curtail disease.1. Altman, D. R. et al. Genome Plasticity of agr-defective Staphylococcus aureus during clinical infection. Infect. Immun.(2018).Disclosures All authors: No reported disclosures.

Improved characterisation of MRSA transmission using within-host bacterial sequence diversity.

Methicillin-resistant Staphylococcus aureus (MRSA) transmission in the hospital setting has been a frequent subject of investigation using bacterial genomes, but previous approaches have not yet fully utilised the extra deductive power provided when multiple pathogen samples are acquired from each host. Here, we used a large dataset of MRSA sequences from multiply-sampled patients to reconstruct colonisation of individuals in a high-transmission setting in a hospital in Thailand. We reconstructed transmission trees for MRSA. We also investigated transmission between anatomical sites on the same individual, finding that this either occurs repeatedly or involves a wide transmission bottleneck. We examined the between-subject bottleneck, finding considerable variation in the amount of diversity transmitted. Finally, we compared our approach to the simpler method of identifying transmission pairs using single nucleotide polymorphism (SNP) counts. This suggested that the optimum threshold for identifying a pair is 39 SNPs, if sensitivities and specificities are equally weighted.

Clostridioides difficile Whole-Genome Sequencing Reveals Limited Within-Host Genetic Diversity in a Pediatric Cohort.

Whole-genome sequencing (WGS) is a highly sensitive method for identifying genetic relatedness and transmission of Clostridioides difficile strains. Previous studies suggest that as few as 3 core genome single-nucleotide variants (SNVs) discriminate between genetically distinct isolates. Because a single C. difficile colony is selected from culture for WGS, significant within-host genetic diversity could preclude identification of transmission events. To evaluate the likelihood of missed transmission events using WGS of single colonies from culture, we examined within-host genetic diversity among C. difficile isolates collected from children. We performed WGS using an Illumina MiSeq instrument on 8 C. difficile colonies randomly selected from each culture performed on stool collected from 10 children (8 children diagnosed with C. difficile infection and 2 children with asymptomatic carriage); 77/80 (96%) isolate sequences were successfully assembled. Among 8/10 (80%) children, all isolates were the same sequence type (ST). The other 2 children each had mixed infection with two STs, although one ST predominated. Among 9/10 (90%) children, isotypic isolates differed by ≤2 SNVs; an isotypic isolate in the remaining child differed by 3 to SNVs relative to the other isolates from that child. Overall, among the 77 isolates collected from 10 stool cultures, 74/77 (96%) were clonal (i.e., same ST and ≤2 core genome SNVs) to other isolates in stool culture. In summary, we identified rare C. difficile within-host genetic diversity in children, suggesting that WGS of a single colony from stool is likely to appropriately characterize isolate clonality and putative transmission events in the majority of cases.

Direct transmission of within-host Mycobacterium tuberculosis diversity to secondary cases can lead to variable between-host heterogeneity without de novo mutation: A genomic investigation.

BackgroundWhole genome sequencing (WGS) has enabled the development of new approaches to track Mycobacterium tuberculosis (Mtb) transmission between tuberculosis (TB) cases but its utility may be challenged by the discovery that Mtb diversifies within hosts. Nevertheless, there is limited data on the presence and degree of within-host evolution.MethodsWe profiled a well-documented Mtb transmission cluster with three pulmonary TB cases to investigate within-host evolution and describe its impact on recent transmission estimates. We used deep sequencing to track minority allele frequencies (<50·0% abundance) during transmission and standard treatment.FindingsPre-treatment (n = 3) and serial samples collected over 2 months of antibiotic treatment (n = 16) from all three cases were analysed. Consistent with the epidemiological data, zero fixed SNP separated all genomes. However, we identified six subclones between the three cases with an allele frequency ranging from 35·0% to 100·0% across sampling intervals. Five subclones were identified within the index case pre-treatment and shared with one secondary case, while only the dominant clone was observed in the other secondary case. By tracking the frequency of these heterogeneous alleles over the two-month therapy, we observed distinct signatures of drift and negative selection, but limited evidence for de novo mutations, even under drug pressure.InterpretationWe document within-host Mtb diversity in an index case, which led to transmission of minority alleles to a secondary case. Incorporating data on heterogeneous alleles may refine our understanding of Mtb transmission dynamics. However, more evidence is needed on the role of transmission bottleneck on observed heterogeneity between cases.