key: cord-0315170-6mhx498c
authors: Cancino-Munoz, I.; Valencia Region Tuberculosis Working Group,; Lopez, M. G.; Torres-Puente, M.; Villamayor, L. M.; Borras, R.; Borras-Manez, M.; Bosque, M.; Camarena, J. J.; Colijn, C.; Colomer-Roig, E.; Colomina, J.; Escribano, I.; Esparcia-Rodriguez, O.; Garcia-Garcia, F.; Gil-Brusola, A.; Gimeno, C.; Gimeno-Gascon, A.; Gomila-Sard, B.; Gonzales-Granda, D.; Gonzalo-Jimenez, N.; Guna-Serrano, M. R.; Lopez-Hontangas, J. L.; Martin-Gonzalez, C.; Moreno-Munoz, R.; Navarro, D.; Navarro, M.; Orta, N.; Perez, E.; Prat, J.; Rodriguez, J. C.; Ruiz-Garcia, M. M.; Vanaclocha, H.; Comas, I.
title: Population-based sequencing of Mycobacterium tuberculosis reveals how current population dynamics are shaped by past epidemics
date: 2022-01-25
journal: nan
DOI: 10.1101/2022.01.24.22269736
sha: dbbcd38e964808ae43914fa716c0ed5830a84b3a
doc_id: 315170
cord_uid: 6mhx498c

Background. Transmission has been proposed as a driver of tuberculosis (TB) epidemics in high-burden regions, with negligible impact in low-burden areas. Genomic epidemiology can greatly help to quantify transmission in different settings but the lack of whole genome sequencing population-based studies has hampered its use to compare transmission dynamics and contribution across settings. Methods. We generated an additional population-based sequencing dataset from Valencia Region, a low burden setting, and compared it with available datasets from different TB settings to reveal heterogeneity of transmission dynamics and its public health implications. We sequenced the whole genome of 785 M. tuberculosis strains and linked genomes to patient epidemiological data. We applied a pairwise distance clustering approach and phylodynamics methods to characterize transmission events over the last 150 years, in Valencia, Spain (low burden), Oxfordshire, United Kingdom (low burden) and a high-burden (Karonga, Malawi). Results. Our results revealed high local transmission in the Valencia Region (47.4% clustering), in contrast to Oxfordshire (27% clustering), and similar to a high-burden setting like Malawi (49.8% clustering). By modelling times of the transmission events, we observed that settings with high transmission are associated with uninterrupted transmission of strains over decades, irrespective of burden. Conclusions. Our results underscore significant differences in transmission between TB settings even with similar burdens, reveal the role of past epidemic in on-going TB epidemic and highlight the need for in-depth characterization of transmission dynamics and specifically-tailored TB control strategies. Funding. European Research Council under the European Unions Horizon 2020 research and innovation program (Grants 638553-TB-ACCELERATE, 101001038-TB-RECONNECT), and Ministerio de Ciencia e Innovacion (Spanish Government, SAF2016-77346-R and PID2019-104477RB-I00)

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The copyright holder for this preprint this version posted January 25, 2022. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Abstract Background. Transmission has been proposed as a driver of tuberculosis (TB) epidemics in high-burden regions, with negligible impact in low-burden areas. Genomic epidemiology can greatly help to quantify transmission in different settings but the lack of whole genome sequencing population-based studies has hampered its use to compare transmission dynamics and contribution across settings.

We generated an additional population-based sequencing dataset from Valencia Region, a low burden setting, and compared it with available datasets from different TB settings to reveal heterogeneity of transmission dynamics and its public health implications. We sequenced the whole genome of 785 M. tuberculosis strains and linked genomes to patient epidemiological data. We applied a pairwise distance clustering approach and phylodynamics methods to characterize transmission events over the last 150 years, in Valencia, Spain (low burden), Oxfordshire, United Kingdom (low burden) and a high-burden (Karonga, Malawi).

Our results revealed high local transmission in the Valencia Region (47.4% clustering), in contrast to Oxfordshire (27% clustering), and similar to a high-burden setting like Malawi (49.8% clustering). By modelling times of the transmission events, we observed that settings with high transmission are associated with uninterrupted transmission of strains over decades, irrespective of burden.

Our results underscore significant differences in transmission between TB settings even with similar burdens, reveal the role of past epidemic in on-going TB epidemic and highlight the need for in-depth characterization of transmission dynamics and specifically-tailored TB control strategies. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The copyright holder for this preprint this version posted January 25, 2022.

Tuberculosis (TB) is one of the top 10 most deadly infectious diseases according to the World Health Organization (WHO). In 2019 were reported 10 million new TB cases and 1.4 million deaths, with these numbers likely to increase due to the COVID-19 pandemic (Glaziou, 2020) . Recognizing heterogeneity across settings in the population-level dynamics of tuberculosis is key to advance to new stages in local and global TB control (Mathema et al., 2017) . Recent transmission significantly contributes to the global TBburden mostly in the high incidence regions and its control is imperative to achieve the goal of the End TB Strategy (Guerra-Assunção et al., 2015; "The transmission of Mycobacterium tuberculosis in high burden settings," 2016). On the contrary, in many countries close to the pre-elimination phase (<5/100,000 cases) ongoing transmission plays a minor role and control strategies focused on latent TB infection (LTBI) mostly from imported cases (Menzies et al., 2018) . However, whether burden can be used as a proxy of recent transmission is not clear and understanding transmission dynamics for each country is key for tailor-made strategies.

Measuring transmission is still challenging, it can be achieved by comparing the pathogen genomes of culture positive cases with some limitations, for example all transmission cases associated with LTBI or those with negative culture cannot be analyzed. However, it allows us to compare transmission clustering rates across countries in a standard way. Whole-genome sequencing (WGS) represents a widely applied tool in the study of TB epidemiology and transmission based on the pairwise single nucleotide polymorphisms (SNPs) distance (Gardy et al., 2011; "Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study," 2013). WGS displays higher resolution, provides accurate results CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 tracking recent transmission ("Aiming for zero tuberculosis transmission in low-burden countries," 2017, "Role and value of whole genome sequencing in studying tuberculosis transmission," Jajou et al., 2018; Meehan et al., 2019) and reports greater agreement with epidemiological results (Nikolayevskyy et al., 2016; Roetzer et al., 2013) . Despite WGS reliability, there exists controversy regarding the SNP threshold employed to delineate genomic clusters. A cut-off of 5 SNPs has been widely accepted for the clustering of recently linked cases (Meehan et al., 2019;  "Role and value of whole genome sequencing in studying tuberculosis transmission," 2019) while an upper value of 12 SNPs also incorporates older transmission events ("Whole-genome sequencing to delineate Mycobacterium tuberculosis outbreaks: a retrospective observational study," 2013); however, the extent to which the identification of those cases can aid epidemiological investigations remains controversial (Bjorn-Mortensen et al., 2016; Jajou et al., 2018) . It is also unclear the extent to which those cutoffs apply to all settings given differences in social, host and pathogen factors across settings. Even if universal, understanding transmission dynamics goes beyond recent transmission events, which have an actionable value for public health, but that do not capture the long-term dynamics in a population.

The lack of WGS studies at the population level represents the main limitation to the validation of these thresholds across clinical settings and to understand the transmission dynamics in different settings. Here we use available datasets from a low burden setting (Oxfordshire, incidence 8.4 cases per 100,000) and from a high burden setting (Malawi, incidence 87 cases per 100,000) and compare to a newly generated dataset.

Spain is a low-incidence country (9.3/100,000) where the contribution of recent transmission to local TB burden remains largely unknown. We applied WGS to investigate the epidemiology and dynamics of TB transmission in the Valencia Region, CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101/2022.01.24.22269736 doi: medRxiv preprint the fourth most populated region of the country, over three years, and evaluated the general use of an SNP threshold in cluster definition in this particular setting. Compared with similar population-based studies from locations with different TB burdens (Guerra-Assunção et al., 2015; Walker et al., 2014) , transmission in Valencia has a prominent role in the current epidemics, with a genomic clustering rate higher than the other lowburden and closer to high-burden settings. Furthermore, our results demonstrate that current TB incidence in Valencia and Malawi mainly derives from sustained transmission over time, with the majority of the linked cases currently observed coming from long-term transmission chains established around 30 years ago.

We sequenced 77% of the TB culture-positive cases reported between 2014-2016 in Valencia Region (Supplemental Table 1 ). 10 samples were removed as non-MTBC isolates or likely mixed infections (Supplemental figure 1). We identified 6 different lineages(L) circulating in the region (Coll et al., 2014; Stucki et al., 2016) , with L4 the most frequent (92.1%) ( Figure 1A ). Table 2 , reporting the sequenced samples as a representative subset of the total culture-positive cases.

Detailed epidemiological analysis is presented in Supplemental Table 3 , remarkably 63% of all cases were Spanish-born patients, while 30% came from high-incidence countries and 7% from other low-incidence countries. 14% of residents are foreign-born, thereby accounting for a TB incidence of 23.6 vs. 6.9 cases per 100,000 among Spanish-born patients. When observed risk factors, we found that 12.4% of patients CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101/2022.01.24.22269736 doi: medRxiv preprint suffered social exclusion, which was more prevalent among foreign-born patients (OR 3.1, CI 1.9-5.1, p<0.001). Diabetes was present in 10.4% of cases; although this was more prevalent in Spanish-born patients (OR 2.7, CI 1.5-5.4, p<0.001), values were similar to disease prevalence in the general population. 

Classic contact tracing identified 66 epidemiological clusters, including 97 cases, accounting for 12.5% of transmission in the Valencia Region ( Figure 1B ). Spanish-born and foreign-born patients equally formed part of an epidemiological cluster. Considering a pairwise distance threshold of 12 SNPs, we identified 112 genomic clusters, including 331 (42.7%) patients, with clusters including from 2 to 12 cases ( Figure 1C CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101/2022.01.24.22269736 doi: medRxiv preprint Supplemental Table 4 ). Although these clusters included foreign-born patients, Spanishborn patients were more likely part of genomically-linked groups (OR 2, CI 1.44-2.79, p<0.001). In this regard, 42 genomic clusters exclusively comprised Spanish-born patients and 8 included only foreign-born patients. Besides Spanish origin and pulmonary localization of TB (OR 2.5, CI 1.60-3.98, p<0.005), no social or risk factor appeared associated with transmission (Supplemental Table 3 ). In addition, 90% of TB cases in Valencia Region are susceptible to all antibiotics used in treatment, so resistance mutations do not have an impact in the clustering.

We also assessed genomic clusters considering different SNP thresholds, and observed that independently of the cut-off considered, the clustering rate obtained by contact tracing was always lower than the genomic estimates ( Figure 1B) . A high number of genomic links were not detected by epidemiological inspection, while some epidemiological links were not corroborated by any genomic clustering threshold ( Figure   2A ). Comparison of both approaches revealed that only 15.4% of the 331 patients within genomic clusters (12 SNPs) had an identified epidemiological link (Supplemental Results, Supplemental Table 5 ).

We benchmarked WGS as a tool to quantify transmission against contact tracing (Diel et al., 2019) , using the latter as the gold standard (Supplemental Table 6 ). In general, as the SNP threshold decreases, sensitivity diminishes, but specificity and accuracy increase. By a ROC curve, we established 11.5 SNPs as the optimal value for the SNP cut-off that maximizes the agreement between epidemiological investigation and genomic data, and genomic clustering appears as an adequate approach to discriminate transmission, as the area under the curve is higher than 0.9 ( Figure 2B ). Then, we used 12 SNPs threshold to define clusters in the following analyses. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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We calculated the percentage of Spanish-born cases clustered by a range of pairwise distances (0-150 SNPs) and compared with the clustering of local cases in other settings CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101/2022.01.24.22269736 doi: medRxiv preprint distance values. The results strongly suggest that a strict transmission threshold of 12 SNPs (or any other threshold) does not apply to all settings, particularly those with higher transmission burdens ( Figure 3A ) and particularly if we want to understand longterm transmission dynamics.

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Next, we evaluated how old are the genomic clusters identified by the standard 12 SNP threshold. Thus, we inferred the age of the local genomic clusters (gClusters) for the three settings. Dating results of the youngest and the oldest gClusters are summarized in Table 1 , while complete results are detailed in Supplemental Tables 7-9. We can trace gClusters 31 years back from the most recent sample collected for both the Valencia Region and Malawi; however, we only retrieved samples that formed part of gClusters, 19 years before the most recent Oxfordshire sample. The alternative calibration samples included (Supplemental Methods) displayed similar results, thereby allowing comparisons among datasets. Thus many gClusters based on 12 SNP thresholds are beyond the action of public health interventions. In fact, when looking at epidemiological linked cases in the Valencia Region, most of them have a common ancestor less than 10 years before the most recent sample, and the distance between samples typically ranged between 0-4 SNPs, with only one cluster separated by 11 SNPs (Supplemental Table 5 ). While the ROC curve indicated a 12 SNP threshold to capture most CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101/2022.01.24.22269736 doi: medRxiv preprint epidemiological links the reality is that strains linked by more than 5 SNP are beyond the action of public health interventions as they involve too old transmission links. Our results imply that events useful for public health investigations are better captured by a 5 SNP threshold even though some epidemiological links are missing. But the reverse is also true, and more dramatic. Even when using a 5 SNP threshold public health only identifies around 15% of the cases in genomic clusters. This holds true even for pairs of isolates with 0 SNP differences. As seen in high-burden countries, when transmission has a prominent role, many transmission events occur outside the traditional household or work settings. -11 1985 [1972-1996] is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10. 1101 /2022 to avoid the influence of imported genotypes. In the case of Oxfordshire, we identified 14 events between 5-25 yB 2016, with the next transmission event being inferred between 100-150 yB 2016 ( Figure 3B , Supplemental figure 2, Supplemental Table 10 ). Thus, a gap of 75 years occurs between the most recent and the oldest transmission events, explaining why the 12 SNP threshold performs well in this setting as a transmission marker. However, even in this setting, genomic transmission clusters defined by a 12 SNP threshold can be traced back to up to 19 years (Table 1) CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Here, we present the first national population-based study in the Valencia Region. We sequenced the whole genome of a representative proportion of all the TB notified cases that provides an accurate picture of the bacterial population structure, during three years. We exhaustively researched TB transmission linked to local epidemiological data and, by comparing to other settings, highlighted four main characteristics defining dynamics and influence on TB incidence.

(I) Transmission can play a significant role in low-burden countries, especially among local-born patients. The percentage of genomically-linked cases (12 SNPs) of around 43% increases to 47% among the Spanish-born population -being 31% among imported cases-, suggesting that transmission among locally-born patients majorly contributes to burden. Percentages remain high when considering a stricter threshold of 5 SNPs for clustering (35% and 39%, respectively). We found higher transmission in the Valencia Region when compared to other low-burden settings, where clustering ranged between CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 2016), epidemiological links are only identified in 18% of all genomically-clustered cases (Yang et al., 2018) , a similar value to that observed in Valencia (15.4%), despite contact tracing occurring in 78% of cases. In those settings, studies have suggested that contact tracing among close contacts will not have a significant effect on TB incidence at a community level (McCreesh and White, 2018; Surie et al., n.d.) , as transmission associates more with social drivers (Mathema et al., 2017) . This likely explains the lack of agreement between genomic and epidemiologic clusters observed in the Valencia Region (62%) compared to other low-burden settings (Diel et al., 2019; Walker et al., 2014) . CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 between linked and unlinked cases seems arbitrary, as a clear SNP cut-off to delineate genomic transmission could not provide precise results ( Figure 4A ). This contrasts with the results of Oxfordshire, where clustering does not change in the range of 12-150 SNPs ( Figure 4B ). In this sense, the SNP threshold choice used to differentiate transmission from unrelated cases remains challenging even in low-burden settings and provides only tentative information (Meehan et al., 2019) . An in-depth evaluation of clustering is needed to understand the particular transmission dynamics. Furthermore, the Valencia Region and Malawi also display continuous and sustained transmission events over time ( Figure 4C ). Those events outside the genomic transmission clusters likely reflect older contagion chains that still contribute to TB incidence today, as a consequence, clustering is continuous in settings exhibiting this transmission dynamics.

The lack of effective past efforts to halt transmission may represent a plausible explanation. Epidemiological data demonstrates that Spain will likely attain a country profile similar to the UK and other low-burden, high-immigration countries. The higher transmission and the older age of transmission chains likely reflects a situation in which Spain suffered from higher disease incidence for most of the 20 th century, reflecting its lower socioeconomic status than neighboring countries. The current control strategies in place in the Valencia Region meet the WHO's targets to reduce TB, including active case findings of close contacts since the 1990s. Improve TB control has led to a continuous drop in case numbers and to an incidence from 22 to 6.4 in the last 20 years.

By contrast, Oxfordshire displays a bimodal distribution of clustering across pairwise distances, and also lacked transmission events other than those involving 12-SNP genomic clusters (Figure 4) . These results agree with the robust reduction in both disease incidence and transmission that occurred until the beginning of the 1990s in the UK; after that, increased HIV infections, immigration and the emergence of TB drug CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101/2022.01.24.22269736 doi: medRxiv preprint resistance fueled the expansion of the non-eradicated TB (Glaziou et al., 2018) . In accordance with this data, we dated ongoing transmission in Oxfordshire back to 1993.

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The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 Our results underscore a primary role for continuous transmission rather than LTBI reactivation or immigration in fueling TB incidence in the Valencia Region, as occurs in many high-burden settings (Bjorn-Mortensen et al., 2016; Guerra-Assunção et al., 2015; López et al., 2020; Yang et al., 2018) . The opposite scenario occurs in other low-burden countries (Jajou et al., 2018; Walker et al., 2014) where transmission is limited and immigration from high-burden countries, also involving reactivation of the disease, represents the primary driver of incidence. In addition, reported meta-analysis from historical epidemiological studies suggests that, contrary to current assumptions, MTB infection may not be lifelong, and most people are able to clear it (Behr et al., 2019) .

This further suggests that the prevalence of LTBI is much lower than assumed, and most of the TB cases we see today are coming either from recent contagion or imported depending on the TB setting. Our data highlight how low-burden TB locations can entail very distinct scenarios that require specifically-tailored management, and that general TB guidelines should not be applied to all areas based solely on incidence rate (Lönnroth et al., 2015) . Understanding heterogeneities in TB transmission dynamics is essential to define tailor-made interventions to halt transmission with a population-level impact, which is key to reducing the incidence of TB worldwide.

Sample selection and study design 1,388 TB cases were reported between 2014-2016 by the Valencian Regional Public Health Agency (DGSP), 1,019 with positive culture. All the available (785) samples were collected from 18 regional hospitals (Supplemental figure 1) CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10.1101 https://doi.org/10. /2022 microbiological records were obtained from the routine TB surveillance system, for 724 of the total samples. All diagnosed TB-positive patients completed a standardized questionnaire provided by the DGSP. M. tuberculosis structure and clustering analysis were performed with the total sequences. Epidemiological and transmission dynamics analysis were carried on with the samples with available information (724).

Approval for the study was given by the Ethics Committee for Clinical Research from the Valencia Regional Public Health Agency (Comité Ético de Investigación Clínica de la Dirección General de Salud Pública y Centro Superior de Investigación en Salud Pública). Informed consent was waived on the basis that TB detection forms part of the regional compulsory surveillance program of communicable diseases. All personal information was anonymized, and no data allowing patient identification was retained.

Clinical isolates were cultured in Middlebrook 7H11 agar plates supplemented with 10% OADC (Becton-Dickinson) for three weeks at 37°C. After an inactivation step (90 °C, 15 min), DNA was extracted using the cetyl trimethyl ammonium bromide method from a representative sample from each patient (four-time plate scraping). All procedures were conducted in a Biological Safety Level 3 laboratory under WHO protocol recommendations. Sequencing libraries were constructed with a Nextera XT DNA library preparation kit (Illumina, San Diego, CA), following the manufacturer's instructions.

Sequencing was performed using the Illumina MiSeq platform.

Data analysis was carried out following a validated previously-described pipeline (http://tgu.ibv.csic.es/?page_id=1794, (Meehan et al., 2019) . Sequencing reads were trimmed with fastp (Chen et al., 2018) , and kraken software (Wood and Salzberg, 2014) CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted January 25, 2022. ; https://doi.org/10. 1101 /2022 was then used to remove non-Mycobacterium tuberculosis complex (MTBC) reads.

Filtered reads were mapped to an inferred MTBC common ancestor genome (https://doi.org/10.5281/zenodo.3497110) using BWA (Li and Durbin, 2009) . SNPs were called with SAMtools (Li, 2011) and VarScan2 (Koboldt et al., 2012) . GATK HaplotypeCaller (McKenna et al., 2010) was used for calling InDels. SNPs with a minimum of 10 reads (20X) in both strands and minimum base quality of 25 were selected and classified based on their frequency in the sample as fixed (>90%) or low frequency (10-89%). InDels with less than 20X were discarded. SnpEff was used for SNP annotation using the H37Rv annotation reference (AL123456.2). Finally, SNPs falling in genes annotated as PE/PPE/PGRS, 'maturase,' 'phage,' '13E12 repeat family protein'; those located in insertion sequences; those within InDels or in higher density regions (>3 SNPs in 10 bp) were removed due to the uncertainty of mapping. Next, variants were compared with recently published catalogues with validated association between mutations and phenotypic resistance (Ngo and Teo, 2019) in order to predict high-confidence resistance profiles to first-and second-line drugs. Lineages were determined by comparing called SNPs with specific phylogenetic positions established (Coll et al., 2014; Stucki et al., 2016 ). An in-house R script was used to detect mixed infections based on the frequency of lineage-and sublineage-specific positions (López et al., 2020) . Read files were deposited in the European Nucleotide Archive (ENA) under the bioproject numbers PRJEB29604 and PRJEB38719 (Supplemental Table 1 CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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The pairwise SNP distance was computed with the R ape package. Genomic clusters were constructed if the genetic distance between at least two patients' isolates fell below a defined threshold. Cluster monophyly was confirmed in a maximum likelihood tree (50,184 SNPs).

Timed phylogenies were inferred with Beast v2.5.1 (Bouckaert et al., 2014) . Ancient TB DNA (Bos et al., 2014) and samples from a recent Spanish outbreak were included as calibration data. Dating was performed using GTR + GAMMA substitution model, a strict molecular clock model, and a coalescent constant size demographic model, as previously described (López et al., 2020) . Three independent runs of Markov Chain Monte-Carlo length chains of 10 million were performed. Adequate mixing, convergence and sufficient sampling were assessed in Tracer v1.6, after a 10% burn-in.

Transmission events were defined as nodes occurring over time phylogenies (Supplemental figure 6) . The rationale for this approach is based on the assumption that if few pathogen mutations are expected to be observed during a host's infection, as is the case of M. tuberculosis, lineages split only at transmission (Hall et al., 2016) . To estimate the number of local transmission events, all ancestral nodes were counted, including local-born tips occurring within 150 years before 2016. CC-BY 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

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Internal migration and transmission dynamics of tuberculosis in Shanghai, China: an epidemiological, spatial, genomic analysis. Lancet Infect Dis 18:788-795. the author/funder, who has granted medRxiv a license to display the preprint in perpetuity