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

In Canada, tuberculosis disproportionately affects the Inuit, a group of indigenous people inhabiting the Arctic regions. Canada is aiming to eliminate tuberculosis among the Inuit by 2030. One way to help stop transmission and prevent future outbreaks is to trace how and where the disease spreads using DNA sequencing. This information can then be used by public health organizations to identify possible interventions.

Typically, the DNA of the bacterium that causes tuberculosis – Mycobacterium tuberculosis, or Mtb for short – is sequenced 50–100 times and a consensus DNA sequence is then generated for each patient from this data. These consensus DNA sequences are then compared to help piece together who infected whom. Recently, scientists have realized that the bacteria a person is infected with may have different DNA sequences due to people being infected with more than one bacterium or the bacterium developing variations in its genome after the infection. However, current DNA sequencing practices may miss these differences, making it harder to trace how the disease spreads.

Now, Lee et al. show that sequencing the DNA of Mtb from an infected person 500–1000 times (i.e. ∼10-20 times more than usual) makes it easier to detect genetic differences and determine how tuberculosis spreads. This approach, also known as ‘deep sequencing’, was used to analyze DNA samples of Mtb collected from about 50 people during an outbreak of tuberculosis in 2011-2012, which had previously undergone standard DNA sequencing.

This deep sequencing approach identified a ‘super-spreading event’ where one person had likely transmitted tuberculosis to up to 17 others during the outbreak. Lee et al. found that most of these people had visited the same ‘gathering houses’ which are social venues in the community. Implementing targeted public health interventions at these sites may help stop future outbreaks.

To fully understand how useful this method will be for tracking the spread of tuberculosis, deep and routine sequencing will need to be compared against each other in different settings and outbreaks. Furthermore, the approach used in this study may be useful for tracking the transmission of other infectious diseases.

Tuberculosis (TB) in Canada is highest among the Inuit, an Indigenous population with a rate over 300 times that of the non-Indigenous Canadian-born population in 2016 (Inuit Tapiriit Kanatami, 2018). Canada recently set a goal of TB elimination in the Inuit by 2030, (Inuit Tapiriit Kanatami, 2018) which will not be achieved without halting ongoing transmission. Previous studies have used genomic data either alone or in conjunction with classical epidemiology to investigate TB transmission dynamics in the Canadian North, (Tyler et al., 2017; Lee et al., 2015a; Lee et al., 2015b) with the aim of identifying clusters to help guide public health interventions. Thus far, such studies have relied on identifying consensus single nucleotide polymorphisms (cSNPs), consistent with prevailing methodology in this field.

Recent studies suggest that incorporation of within-host diversity into genomic analyses may provide greater resolution of transmission than cSNP-based approaches alone (Worby et al., 2017; Martin et al., 2018; Meehan et al., 2019; Séraphin et al., 2019). This may be particularly important for investigation of outbreaks occurring over short time scales and/or in settings such as the Canadian North, where the genetic diversity of circulating strains is especially low. In both of these circumstances, it is common to find many samples separated by zero cSNPs, hindering accurate source ascertainment. To investigate this hypothesis, we used deep sequencing (i.e., to ~10-20 fold more than standard, or 500-1000x) to re-evaluate transmission in a densely-sampled outbreak in Nunavik, Québec.

This outbreak, which has been previously described, (Lee et al., 2015b; Lee et al., 2016) comprised 50 microbiologically-confirmed cases of TB who were diagnosed in a single Inuit community between 2011–2012 - a rate of 5,359/100,000 for that year. Genomic epidemiology analyses using sequencing depths of ~50x that are standard in such work, identified multiple clusters of transmission in this outbreak, (Lee et al., 2015b) however, there was insufficient genetic variation detected to infer precise person-to-person transmission events within these subgroups, given the short time frame and low mutation rate of M. tuberculosis (~0.2–0.3 SNPs/genome/year for Lineage 4; Menardo et al., 2019). In this study, we illustrate how within-host diversity can be incorporated into transmission analyses. In doing so, we find new features of the transmission networks in this community, in particular, identifying a previously unrecognized super-spreading event. We highlight a potential role for deep sequencing in public health investigations, with implications for TB control in Canada’s North as well as other high-transmission environments.

62/65 (95·4%) available TB samples from cases diagnosed between 2007–2012 were successfully sequenced and passed quality control. This included 48/49 (98·0%) of the samples with an identical Mycobacterial Interspersed Repetitive Units Variable Number Tandem Repeats (MIRU-VNTR) pattern during the outbreak year. The remaining three samples could not be re-grown. Reads that were non-MTBC were removed (Source data 1) and there was no obvious association between percent contamination and hSNP frequency. Epidemiological and clinical data on all outbreak cases are described in Lee et al. (2015b).

Average genome coverage and depth across the H37Rv reference was 98·64% [SD 0·07%] and 714·53 [SD 92·68], respectively. Our primary filtering protocol yielded 51,430 cSNPs and 4,897 hSNPs across all individual samples (Source data 2). Excluding positions that were invariant compared to the reference or where any sample was missing and/or was low-quality resulted in a core alignment of 860 cSNP positions and 136 hSNP positions (note, these are not mutually exclusive, as positions with cSNPs in some samples may have hSNPs in others).

42 positions had hSNPs that were shared across all 62 samples (Table 1, Source data 3). Depth of coverage at these positions was, on average, 39% higher than the average depth across the same sample (SD 36·7%, Source data 4). Along with manual review of alignments (Figure 1), this suggested that many of these were false positives, potentially due to alignment error (e.g., from underlying structural variation in our samples compared to the H37Rv reference).

Figure 1

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To address this, we generated a local reference genome for the outbreak, MT-0080_PB. Quality metrics for the MT-0080_PB assembly are given in Source data 5. Compared to H37Rv, mean genome coverage and depth were higher with MT-0080_PB (at 99·33% [SD 0·09%] and 717·07 [SD 93·01], respectively), fewer positions were missing/low-quality (p < 0·00005, Table 1), and overall, fewer variable positions were detected (p < 0·00005). While core cSNP distances were similar between samples regardless of the reference (Table 1), the number of hSNPs was greatly reduced using MT-0080_PB (Source data 2); while 4,897 hSNPs were identified across all individual samples using H37Rv, only 125 hSNPs were identified using MT-0080_PB. There were also no hSNPs shared across all 62 samples using MT-0080_PB. Together, these findings support our hypothesis that alignment error is responsible for many of the detected variants, and indicate a local reference is important for accurate identification of hSNPs. All further results presented are based on the MT-0080_PB alignment.

A maximum likelihood tree was generated from 90 core cSNP positions (excluding sites invariant across all samples and the reference) compared to MT-0080_PB (Figure 2). All 62 samples (historical and outbreak) were included, for comparison with our previous work. (Lee et al., 2015b) Consistent with this, (Lee et al., 2015b) hierBAPS identified two main sub-lineages (‘Mj-V’ and ‘Mj-III’ per Lee et al., 2015a), with three sub-clusters (Mj-IIIA/B/C).

Figure 2 with 1 supplement see all

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As the TB epidemic continues among the Canadian Inuit, targeted public health interventions are essential to halt ongoing transmission. In order to do so, it is important that transmission events and associated risk factors are accurately identified. Our previous work suggested that hSNP analysis could enhance resolution of TB transmission (Martin et al., 2018). To investigate how this approach could be applied for TB control, we used deep sequencing to re-examine a major TB outbreak in the Canadian Arctic.

Several recent studies, including work by our group (Martin et al., 2018), have shown that M. tuberculosis within-host diversity can be transmitted between individuals (Séraphin et al., 2019; Guthrie et al., 2019). Using deep sequencing data allowed us to better identify this diversity in a Nunavik outbreak compared to previous analyses with standard sequencing depth, (Lee et al., 2015a; Lee et al., 2015b) and facilitated detection of a novel super-spreading event, where one source case may have transmitted to ~1/3 of the other cases diagnosed between 2011 to 2012. This was in addition to a previously identified super-spreader linked to 19 secondary cases - suggesting up to 75% of the outbreak (36/48, excluding the putative super-spreaders) may be attributable to these events. Super-spreading has been described in a number of pathogens, (Stein, 2011) including TB (Kline et al., 1995). Our findings suggest this can play an important role in driving TB outbreaks, and that accurate detection of super-spreading events is important for informing appropriate public health interventions. In the case of MT-504, as nearly all of the secondary cases had attended the same local community gathering houses as the putative source, this strongly suggests these venues play an important role in facilitating transmission in this setting.

Several studies have used genomics to investigate TB recurrence, (Witney et al., 2017; Bryant et al., 2013; Guerra-Assunção et al., 2015) however, the methods used to assess for mixed infection at either time point have been inconsistent and may not be sufficient to discriminate recurrence in settings with low strain diversity. In this analysis, we provide proof-of-principle that deep sequencing can potentially help rule out relapse. The distinction between relapse and re-infection is important at individual and population levels; high rates of relapse in a community would indicate a problem with treatment or adherence, potentially warranting changes to clinical management, while re-infection would indicate the need for public health interventions such as active case finding. Also, individuals in Nunavik who have had prior treatment for active TB disease in the past are also not routinely offered prophylaxis on re-exposure, based on historical data suggesting ~80% protection is afforded by prior infection (Menzies, 1997). The degree to which re-infection drives recurrence in Nunavik is currently unknown, but if re-infection is the primary cause, this clinical practice may need to be re-evaluated. A population-level genomic epidemiology study is currently underway to evaluate this.

To use deep sequencing to investigate within-host diversity, it is critical we minimize false positive hSNPs. We have shown that using a local strain as a reference can not only reduce error, but improves detection of epidemiologically-informative variants. Genomic differences between outbreak strains and H37Rv have been previously illustrated by Roetzer et al. (2013); O'Toole and Gautam (2017), with O'Toole and Gautam (2017) warning that clinical TB strains may be needed to fully detect virulence genes in reference-based analyses. Our analysis suggests these may also be warranted for hSNP analysis; where possible, we suggest using long-read sequencing to generate complete and local reference genomes.

Overall, our study has a number of strengths. Firstly, we had access to a densely-sampled outbreak, which was previously investigated using ‘standard’ sequencing depth and for which detailed epidemiological data was available. This allowed us to readily compare methodological approaches, showing how and when deep sequencing might be beneficial for public health. In this study, we have also identified important methodological considerations for hSNP detection, with implications for transmission analyses, but also potentially for resistance prediction as well (Liu et al., 2015). Finally, the use of long-read data has allowed us to completely assemble a novel TB genome from Nunavik. This will serve as a valuable resource for future studies of transmission in Nunavik (given the low strain diversity in the region Lee et al., 2015a), as well as other Inuit territories.

A potential limitation of this work is that, given the historical nature of the outbreak, deep sequencing was done using DNA extracted from culture. Due to methodological challenges of sequencing directly from sputum, (Brown et al., 2015; Votintseva et al., 2017; Doyle et al., 2018) few studies have examined the effect of culture on genome diversity. A recent study by Shockey et al. (2019)., which analyzed allelic variation among reads from individual samples, suggests that some diversity may be lost during the culturing process. However, several studies looking at potential transmission (Votintseva et al., 2017; Doyle et al., 2018; Nimmo et al., 2019) found results were congruent between cSNP analyses from culture versus raw samples. In terms of hSNPs, Votintseva et al. (2017) Doyle et al. (2018); Nimmo et al. (2019) reported detecting fewer hSNPs with sequencing from culture versus from sputum, in Nimmo et al. (2019), the median hSNPs was only 4.5 versus 5 hSNPs, respectively – a difference that may not be clinically significant, regardless of statistical significance. Given the inconsistency of results and paucity of data, further study is needed to understand how hSNP diversity may be affected by the culturing process, and to assess whether this affects inferences of transmission. We note that it is likely that enhanced detection of the hSNPs present in sputum would improve the resolution over that which we present in this work.

Another potential limitation is that, while we can compare the epidemiological inferences made between our previous analysis and our deep sequencing analysis, the sequencing data and bioinformatics pipelines themselves are not directly comparable. Methods to accurately identify hSNPs and incorporate them into transmission analyses are currently an area of active research. We illustrated in our recent paper (Martin et al., 2018) that additional steps and strict thresholds must be used to minimize false positive hSNPs, and have conducted the current analysis in consideration of this. However, we note that pre-filtering, our 2015 analysis found that MT-504 had five reference alleles at position 276,685 in the H37Rv alignment (out of 75) and randomly downsampling the current data to simulate ~50 x yielded similar results (5/47 reads at position 276,544 aligned to MT-0080_PB). As most genomic studies of TB employ conservative thresholds of 75–90% allele frequency to classify cSNPs, many bioinformatics pipelines would consider this heterogeneity as potentially suspect at standard sequencing depth. This suggests that greater depth and/or different analytic approaches (e.g., Wyllie et al., 2019) are needed to ensure accurate discrimination of sequencing/analytic error from true variation; ultimately, the optimal approach used to identify variants needs to be carefully considered, and appropriate for the study question and data being analyzed.

Finally, while deep sequencing allowed us to detect a novel superspreading event in this context, this approach may not always be necessary; indeed, our previous analysis had identified another super-spreader in the same outbreak using routine sequencing. We acknowledge that this Northern outbreak may not be representative of outbreaks from other settings and/or involving other M. tuberculosis lineages. Further studies are needed to quantify the degree to which super-spreading occurs in TB, and examine how and when deep sequencing should be used to detect this.

In summary, we have found evidence of mixed variants with important epidemiologic implications that would not have been detected with standard methods and common filtering criteria. To our knowledge, however, no other studies have been published comparing epidemiological inferences obtained with deep versus routine sequencing for TB outbreak resolution – thus this work represents an important methodological advance in this area. We illustrate that genomic methods, while powerful, still require careful interpretation and can still harbor considerable ambiguity when comparing very close links in a transmission chain, or, as also suggested in Xu et al. (2019), when trying to identify source cases. This finding is likely relevant beyond TB, given the increasing number of pathogens undergoing genomic investigation. Our work also highlights the importance of reproducing previous genomic epidemiology analyses; as the technology and methods used in this field continue to develop, these can lead to improved resolution of transmission and ultimately, challenge previously-held inferences – with critical implications for public health. In terms of TB control, we demonstrate that deep sequencing can aid in transmission analyses, in particular by allowing accurate identification of TB super-spreading events and associated epidemiological characteristics. We propose that deep sequencing is most useful for understanding transmission in settings with low strain diversity, and that these may benefit from routine use of this approach. We hypothesize deep sequencing may also provide additional resolution of transmission events within outbreaks occurring over short, limited timescales – irrespective of local strain diversity, as (by definition) all samples involved in recent transmission would be expected to be closely-related. However, further studies comparing deep versus routine sequencing, ideally from a diversity of clusters and epidemiologic contexts, are needed to fully quantify the added value of this approach for epidemiologic studies of TB.

Overall, this work has important implications for the Canadian North, as well as other regions with high TB transmission; as next-generation sequencing becomes a mainstay in public health surveillance, it is critical we recognize the limitations of analyses done using routine sequencing data. Accurate resolution of transmission is essential for TB control programmes, in order to better understand risk factors for such transmission and enable prioritization of public health resources. With respect to Nunavik, our findings were regarded as very valuable by the regional public health unit and local community leaders; as a direct consequence of this work, ongoing and new investigations of TB genomic epidemiology in Nunavik are using deep sequencing to inform transmission analyses. However, while costs continue to decline, we recognize deep sequencing of all samples in an outbreak may not be economically viable in every setting.

Sequencing data and the assembly for MT-0080 are available on the NCBI's Sequence Read Archive under BioProject PRJNA549270.

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Author details

1. Robyn S Lee

   1. Epidemiology Division, Dalla Lana School of Public Health, University of Toronto, Toronto, Canada
   2. Center for Communicable Disease Dynamics, Harvard TH Chan School of Public Health, Boston, United States
   3. Department of Epidemiology, Harvard TH Chan School of Public Health, Boston, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration

   For correspondence

   robyn.s.c.lee@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7120-9053

2. Jean-François Proulx

   Nunavik Regional Board of Health and Social Services, Kuujjuaq, Canada

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

3. Fiona McIntosh

   The Research Institute of McGill University Health Centre, Montréal, Canada

   Contribution

   Resources

   Competing interests

   No competing interests declared

4. Marcel A Behr

   The Research Institute of McGill University Health Centre, Montréal, Canada

   Contribution

   Resources

   Competing interests

   No competing interests declared

5. William P Hanage

   1. Center for Communicable Disease Dynamics, Harvard TH Chan School of Public Health, Boston, United States
   2. Department of Epidemiology, Harvard TH Chan School of Public Health, Boston, United States

   Contribution

   Conceptualization, Resources, Supervision, Funding acquisition, Methodology

   Competing interests

   No competing interests declared

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