Science of the Total Environment 843 (2022) 156970

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Detection of M. tuberculosis in the environment as a tool for identifying

high-risk locations for tuberculosis transmission
⁎
Renu Verma a, , Flora Martinez Figueira Moreira f, Agne Oliveira do Prado Morais d, Katharine S. Walter a,
Paulo César Pereira dos Santos d, Eugene Kim a, Thiego Ramon Soares b, Rafaele Carla Pivetta de Araujo b,
Bruna Oliveira da Silva b, Andrea da Silva Santos b, Julio Croda c,e,f, Jason R. Andrews a
a
Division of Infectious Diseases and Geographic Medicine, Stanford University School of Medicine, Stanford, CA, USA
b
Faculty of Health Sciences, Federal University of Grande Dourados, Dourados, MS, Brazil
c
Oswaldo Cruz Foundation Mato Grosso do Sul, Mato Grosso do Sul, Brazil
d
Postgraduate Program in Infectious and Parasitic Diseases, Federal University of Mato Grosso do Sul, Mato Grosso do Sul, Brazil
e
Department of Epidemiology of Microbial Diseases, Yale School of Public Health, New Haven, USA
f
Federal University of Mato Grosso do Sul - UFMS, Campo Grande, MS, Brazil

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• M. tuberculosis was detectable by PCR on

room surfaces where TB cases were pres-
ent.
• Detection of Mtb on surfaces correlated
with patients' sputum bacillary load.
• Using RNA baits, we captured and se-
quenced Mtb directly from environmental
swabs.
• We identified genomic markers of antibi-
otic resistance in Mtb from surface swabs.

A R T I C L E I N F O A B S T R A C T

Editor: Ewa Korzeniewska Tuberculosis (TB) remains a leading cause of infectious mortality globally, yet most cases cannot be epidemiologically
linked even with extensive contact investigations and whole genome sequencing. Consequently, there remain major
Keywords: gaps in our understanding of where and when M. tuberculosis (Mtb) exposures occur. We aimed to investigate whether
Mycobacterium tuberculosis Mtb can be detected in environments where TB patients were recently present, which could serve as a tool for charac-
Environment
terizing exposure risk. We collected 389 environment surface (ES) swabs from two high TB burden prisons in Brazil,
Transmission
Whole genome sequencing
sampling 41 (n = 340) cells occupied by individuals with active TB and 7 (n = 49) cells from individuals without
TB. In a subset of pooled swabs (n = 6) and a swab from a cigarette lighter from the cell with active TB patients,
we enriched Mtb DNA using RNA-bait hybrid capture assays and performed whole genome sequencing. In prison
cells, Mtb DNA was detected in 55/340 (16 %) of ES swabs from cells occupied by active TB patients and none (0/
49) from cells in which no active TB patients were present. Mtb was detected in 13/16 (81 %) prison cells occupied
by the individuals with high/medium sputum Xpert Mtb load and 8/25 (32 %) with low/very low sputum Mtb load
(p = 0.003). Seven hybrid capture samples had a median genomic coverage of 140×. rpoB mutations conferring
high-level rifampin resistance were detected in 3/7 ES swabs. Mtb was frequently detectable in environments recently
occupied by individuals with active TB. This approach could be applied in congregate environments to identify and
characterize high-risk settings for Mtb exposure.

⁎ Corresponding author at: Biomedical Innovations Building, Room 3600, 240 Pasteur Drive, Stanford, CA 94305, USA.
E-mail address: vrenu@stanford.edu (R. Verma).

http://dx.doi.org/10.1016/j.scitotenv.2022.156970
Received 26 April 2022; Received in revised form 21 June 2022; Accepted 21 June 2022
Available online 26 June 2022
0048-9697/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
R. Verma et al.
1. Introduction
Despite the availability of highly effective treatment regimens and
reliable rapid diagnostics, >10 million new cases of TB are estimated
to occur every year (Global tuberculosis report, 2021). A major obstacle
to reducing this burden is ongoing transmission of infection. Classical
TB transmission studies predominantly rely on detecting Mtb either
from patient-derived samples such as sputum or bioaerosols generated
by an active TB patient at a single time-point (Ragonnet et al., 2019;
Rufino de Sousa et al., 2020a; Patterson et al., 2020). However, a
major limitation of this approach is that infectious individuals remain
undiagnosed for several weeks to months, making it challenging to iden-
tify locations of transmission (Wood et al., 2007). Using molecular data
combined with household and social contacts, studies have reported
that only a minority of Mtb infections in adults are attributable to
these identifiable contacts (Surie et al., 2017; McCreesh and White,
2018; Andrews et al., 2014). A study in KwaZulu-Natal in South Africa
found that only 12 % of the transmission was molecularly linked to
close contacts (Wilkinson et al., 1997). In another HIV-prevalence
community in South Africa, only 19 % of clustered TB cases could be
attributed to household transmission (Middelkoop et al., 2015). These
challenges have also been independently observed in rural Malawi
where only 9 % of TB cases were attributable to known contacts
(Glynn et al., 2015). Taken together, these findings suggest that most
Mtb infections are acquired in congregate settings outside the house-
hold; however, there is little direct evidence about where such transmis-
sion occurs.
Several emerging approaches have been undertaken in attempts to
identify high risk locations for Mtb exposure in community settings. These
include social network analysis among individuals diagnosed with TB
(Packer et al., 2019), use of wearable GPS loggers to identify durations
individuals spend in various environments (Rainham et al., 2008), and
mapping of ‘rebreathed air’ across communal environments using portable
carbon dioxide monitors (Wood et al., 2014). These approaches provide
insights into potential exposure risks, though such evidence is indirect in
the absence of Mtb detection in these environments.
We hypothesized that Mtb DNA could be detectable on surfaces in
environments where active TB patients were recently present, providing
direct evidence about exposure risk in these environments. To test this
hypothesis, we developed an environmental sampling and molecular
testing approach for Mtb DNA detection and evaluated its application
in a very high-risk environment–prisons. We further tested whether
we could recover full Mtb genome sequences from environmental
samples.
2. Methods
2.1. Ethics statement
The clinical study was approved by the institutional review boards of
the Federal University of Grande Dourados, Brazil (IRB#3.483.377) and
Stanford University (IRB# 40285). All participants were over 18 years of
age and provided written informed consent to participate.
2.2. Tuberculosis screening in prisons
As a part of an active case finding program, our team performed mass
screening for TB in two prisons in Brazil (Penitenciária Estadual de
Dourados and Estabelecimento Penal Jair Ferreira de Carvalho) by
chest radiography and sputum testing by GeneXpert MTB/RIF Ultra
assay and culture (Santos et al., 2021). All incarcerated individuals
were offered screening and > 90 % agreed to participate. Sputum testing
was performed for all individuals who were able to produce sputum,
irrespective of reported symptoms. Based on these data, we identified
41 cells occupied by active TB patients, in which we undertook environ-
mental surface sampling.
2
Science of the Total Environment 843 (2022) 156970
2.3. Assay development and validation
For assay development and validation, we collected ES swabs from three
negative pressure TB isolation rooms at the time of patient discharge. We de-
tected Mtb in 44 % (19/43) swabs from the two rooms with smear positive
patients and none (0/33) of the swabs collected from a room with a smear-
negative TB patient. We found good concordance in positivity in paired
swabs assayed by in-house real-time PCR and GeneXpert (Supplemental
methods and results). A complete list of surfaces which were swabbed for
Mtb DNA detection is provided in Supplementary Table 1.
Sterile Hydraflock swabs (Longhorn Primeswab LH-11-10) were used to
collect ES swabs. Surfaces covering an area roughly the size of an adult
palm (approx. 70 cm2) were swabbed with a hyraflock swab that was pre-
wet with buffer (1× TE or 1× PBS). After collection, the ES swabs were
immediately dispensed in 500ul buffer and transported to the molecular
lab. We tested the Mtb DNA detection methods on two different platforms:
GeneXpert MTB/RIF Ultra (Cepheid, Sunnyvale, USA) and StepOnePlus
real time PCR machine (Applied Biosystems) using insertion sequence
(IS) 6110 and the region of difference 9 (RD-9) custom TaqMan assays
described previsouly (Huard et al., 2003). IS6110 is typically present in
multiple, but variable numbers of copies in the Mtb genome, making it a
sensitive target for detection but precluding quantification of genome
copies, whereas RD-9 is present as a single copy, enabling genome copy
quantification.
2.3.1. Custom TaqMan assays
For custom TaqMan assays, we collected ES swabs in 500ul 1× TE
buffer (pH = 7.4) (Millipore Sigma) for DNA extraction. DNA was
extracted using QIAamp DNA mini kit (Cat # 51304). All qPCR reactions
were performed in 20ul volume using TaqMan™ Environmental Master
Mix 2.0 (Cat# 4396838) with 500 nM of each forward and reverse primers
and 250 nM of probes. The samples were analyzed in technical duplicates
on a StepOnePlus real time PCR using the following cycling conditions;
initial denaturation (95 °C;10 min), 40 cycles (denaturation 95 °C 15 s
and 60 °C 30 s annealing and extension). The samples were considered
positive only when the amplification was observed in both duplicates of
IS6110 and RD-9 assays. DNA copies were quantified using a RD-9 standard
curve derived from Mtb H37Rv ATCC strain (Catalogue#25618DQ)
(Supplementary Fig. 1).
2.3.2. Analysis on GeneXpert MTB/RIF Ultra
For GeneXpert analysis, we collected ES swabs in 500ul sterile 1× PBS
(pH = 7.4) (Cytiva HyClone™) and used directly without manual DNA
extraction. Briefly, the swabs were vortexed for 30 s and the samples
were mixed with Xpert MTB/RIF buffer in 1:1 (sample to buffer ratio).
The samples were vortexed for 5 s followed by 5 min incubation at room
temperature. The entire 1 ml sample was loaded into GeneXpert MTB/RIF
Ultra cartridge for Mtb DNA detection. GeneXpert Mtb load was graded
semiquantitatively as high (Ct <16), medium (Ct 16 < 22), low (Ct
22–28), very low (Ct >28) and trace).
2.4. Environmental surface sampling in prison cells
We collected 340 swabs from prison cells (n = 41) where an individual
active TB was diagnosed within the past 14 days (Table 1), 49 swabs from
cells (n = 7) where no active TB patient had been recently diagnosed, and
70 ES swabs from low-risk sites, including residential apartments (n =
7) and a non-TB lab (n = 1) at the Federal University of Grande Dourados,
Brazil. For security reasons, the ES swab samples were collected by the
incarcerated individuals who were provided brief instructions by the
study staff. Prior swab collection, study staff briefly explained the purpose
and importance of collecting ES swabs from the prison cells followed by a
demonstration on the swabbing technique and dispensing the swab in
the collection tube. The study staff waited outside the cell instructing the
incarcerated individuals about which surfaces they should swab (Supple-
mentary Fig. 2). The swabs were collected in five categories- clothing,
R. Verma et al.
bedding, bathroom, personal utensils, and other surfaces in the cell which
included floors, windows, walls, ceilings, and doors (Table 2).
2.5. Sample collection for whole genome sequencing
Based on the GeneXpert results on ES swabs, we identified seven cells
with highest rates of Mtb DNA positivity and collected additional 85 ES
swabs for DNA enrichment with RNA baits-based hybrid capture assays
and whole genome sequencing. The swabs were collected in 500ul 1×
TE buffer. We first processed 15 swabs randomly chosen from a batch of
85 samples and individually analyzed those on IS6110 TaqMan assay.
Only one swab collected from a cigarette lighter which was earlier positive
on GeneXpert MTB/RIF Ultra was positive on the custom TaqMan assay.
Therefore, to maximize the chances of detecting Mtb DNA, instead of ana-
lyzing the swabs individually, we divided the swabs from each cell into
three categories- utensils, bed & clothing and other surfaces. We pooled
the samples within each category before DNA elution to get total three
swabs (one from each category) per cell. DNA was extracted as described
above and eluted in 30ul nuclease-free water.
2.6. Mtb culture from paired sputum samples
To compare the enriched genomes of ES swab samples with the
genomes of Mtb present in the sputum of individuals occupied those cells
at the time of swab sampling, we further cultured Mtb from three sputum
samples using Ogawa-Kudoh Method described previously (Costa et al.,
2018). Briefly, a sterile cotton swab was dipped and swirled in 2 ml sputum
and submerged into a 4 % solution of sodium hydroxide (NaOH) for 2 min
to decontaminate the sample. The excess of 4 % NaOH was removed by
compression of the swab against the flask wall and the sample was inocu-
lated in Ogawa-Kudoh medium (pH 6.4). The cultures were incubated at
37 °C and observed for growth twice a week for 60 days. Mtb DNA was
extracted using Cetyltrimethyl ammonium bromide (CTAB) method
described previously (Huard et al., 2003).
2.7. Hybrid capture assays and whole genome sequencing
We performed IS6110 TaqMan qPCR on DNA extracted from 21 pooled
swabs corresponding to seven cells. Six pooled samples from three cells and
one swab from a cigarette lighter, all positive on IS6110 assay, were consid-
ered for hybrid capture assays. DNA from ATCC H37Rv was used as a pos-
itive control. Because of the relatively low quantity of Mtb DNA in ES swab
samples compared to routinely sequenced cultured sputum, we took an
RNA hybrid capture approach, allowing us to selectively enrich for Mtb
DNA from a mixed template (Goig et al., 2020). Six pooled swabs including
three from utensils, two from bed and clothing, and 1 from other surfaces in
addition to one swab from a cigarette lighter corresponding to three cells
were positive on IS6110 qPCR and were selected for hybrid capture and
whole genome sequencing (Table 3). To compare captured ES swab
samples to sequenced cultured sputum, we additionally sequenced three
cultured sputum samples from the Mtb positive individual in the same cells.
All sequencing libraries were prepared using Illumina DNA Prep library
preparation kit (Cat # 20018704) following manufacturer instructions.
Table 1
Characteristics of individuals with active tuberculosis in study prison cells selected
for environmental sampling.
Sampling sites Prison EPJFC Prison PED Total (n)
Number of cells sampled 26 15 41
Individuals with active TB 28 17 45
Individuals with cough 13/28 (46 %) 9/17 (53 %) 22/45 (49 %)
Bacillary load in sputum samples based on GeneXpert grading*
High 6 (21 %) 8 (47 %) 14 (31 %)
Medium 5 (18 %) 1 (6 %) 6 (13 %)
Low 8 (29 %) 6 (35 %) 14 (31 %)
Very low 9 (32 %) 2 (12 %) 11 (24 %)
3
Science of the Total Environment 843 (2022) 156970
Table 2
Detection of Mtb DNA on environmental surface swabs from prison cells.
ES swab sampling sites
Prison EPJFC Prison PED Total (n)
Number of prison cells sampled 26 15 41
Surfaces swabbed 189 151 340
Swabs positive on GeneXpert 19/119 (16 %) 30/151 (20 %) 49/270 (18 %)
Swabs positive on StepOne Plus 6/70 (9 %) – 6/70 (9 %)
Total Positive 25/189 (13 %) 30/151 (20 %) 55/340 (16 %)
Following library preparation, we pooled eight libraries consisting of
seven samples from prison cells and one with H37Rv DNA (ATCC; 104 cop-
ies). To achieve maximum genome coverage in hybrid capture reactions,
we split the libraries into 4 parts (7ul each) and enriched each library
using biotinylated RNA-baits (myBaits®; Arbor Biosciences) based on the
inferred ancestral genome of the Mtb complex, which is genetically equidis-
tant to all Mtb lineages. We performed the baits protocol for four replicate
reactions following the standard protocol published by the manufacturer
using a hybridization and wash temperature of 60 °C. Briefly, the concen-
trated library pools were first split into four reactions, each undergoing
hybridization incubation (60 °C, 24 h). These reactions were then bound
to streptavidin-coated magnetic beads and washed (60 °C). Finally, each re-
action was split into two 15uL aliquots and performed a post-capture PCR
amplification of both aliquots (8 total PCR reactions) using 2× KAPA
HiFi HotStart Ready Mix (Roche) and 10× KAPA Library Quantification
Primer Mix (Roche). The post-capture amplification reaction consisted
of an initial denaturation step at 98 °C (2 min), then 12 cycles of 98 °C
(20 s), 60 °C (30 s), and 72 °C (45 s). Final extension was at 72 °C (5 min)
and held at 8 °C until 0.8× Agencourt Ampure SPRI beads cleanup. A
total 20uL of cleaned enrichments were analyzed on the Illumina Miseq
in paired-end runs of 2x292bp length.
2.8. Whole genome sequencing and bioinformatic methods
We trimmed low-quality bases (Phred-scaled base quality <20) and re-
moved adapters with Trim Galore (stringency = 3) (Krueger, 2019). We
used CutAdapt to further filter reads (−-nextseq-trim = 20 –minimum-
length = 20 –pair-filter = any) (Kechin et al., 2017).To exclude potential
contamination, we used Kraken2 to taxonomically classify reads and re-
moved reads that were not assigned to the Mycobacterium genus or that
were assigned to a Mycobacterium species other than Mtb (Wood and
Salzberg, 2014). We mapped reads with bwa v. 0.7.15 (bwa mem) (Li and
Durbin, 2009) to the H37Rv reference genome (NCBI Accession: NC_
000962.3) and removed duplicates with sambamba (Tarasov et al., 2015).
We called variants with GATK 4.1 HaplotypeCaller (Van der Auwera and
O'Connor, 2020), setting sample ploidy to 1, and GenotypeGVCFs, including
non-variant sites in output VCF files. We included variant sites with a mini-
mum depth of 10× and a minimum variant quality score 40 and constructed
consensus sequences with bcftools consensus (Danecek et al., 2021), exclud-
ing indels, and used SNP-sites to extract a multiple alignment of SNPs
between sampled genomes (Leaché et al., 2015). We excluded SNPs in previ-
ously defined repetitive regions (PPE and PE-PGRS genes, phages, insertion
sequences and repeats longer than 50 bp) (Brites et al., 2018). We measured
drug resistance associated mutations with MykrobePredictor v0.8.0, using
the ‘201901’ database of genomic predictors of resistance (Bradley et al.,
2015a). We identified lineage and drug resistance associated mutations
with TBProfiler v.2.8.6 (Phelan et al., 2019). Our bioinformatic pipeline is
available at https://github.com/ksw9/mtb_pipeline. Sequence data is avail-
able at the Sequence Read Archive, BioProject ID PRJNA846807.
2.9. Phylogenetic analysis
We used the R package ape to measure the number of pairwise SNP
differences between samples (Li and Durbin, 2009). Missing sites, “Ns” in
the alignment, were not counted as pairwise differences. To explore if
R. Verma et al.
Table 3
Hybrid capture assay and sequencing statistics for environment samples and samples extracted from matched sputum cultures.
Cell Sample category Lineages detected Median coverage (X)
1 Sputum culture 4.3.4.2 51
1 Bed & clothing 4.8; 4.1.2.1 81
1 Utensils 4.8; 4.1.2.1 176
1 Other Surfaces 4.8; 4.1.2.1 103
2 Sputum culture 4.1.2.1 48
2 Bed & clothing 4.1.2.1 132
2 Utensils 4.1.2.1 21
2 Lighter 4.1.2.1 215
3 Sputum culture 4.1.2.1; 4.1.1.3 60
3 Bed & clothing 4.1.2.1 217
heterozygous calls in samples with mixed infections were contributing to
observed pairwise distances between samples, we excluded heterozygous
sites with a minor allele frequency > 5 % using bcftools and recalculated
pairwise SNP distances.
We fit maximum likelihood trees with RAxML-ng 1.0.1 and used a GTR
substitution model and a Stamatakis ascertainment bias correction for
invariant sites in our alignment. We divided the number of invariant sites
by 1000 to avoid issues created by small branch lengths. We defined nucle-
otide stationary frequencies as frequencies in the reference genome.
2.10. Data analysis
We compared the detection of at least one swab in a cell by GeneXpert
semi-quantitative grade using Fisher's exact test. We used mixed effects
logistic regression with random effect for cell to assess the relationship
between Xpert semiquantitative grade and swab positivity. All statistical
analyses were performed using R (R Core Team, 2018).
3. Results
3.1. Mtb DNA detection in prison cells
We sampled 48 prison cells, 41 of which were occupied by 45 active TB
patients. At any time point during swab collection, each cell was occupied
with multiple individuals without TB sharing a cell with active TB patients
(median = 14; IQR, 11–17). Thirty-seven cells housed one active TB pa-
tient each, whereas four cells accommodated two active patients. Approxi-
mately half (22/45; 49 %) of participants with diagnosed TB reported
cough at the time of swab collection. At the time of diagnosis, sputum
GeneXpert semi-quantitative grade was high in 14/45 (31 %) participants,
medium in 6/45 (13 %), low in 14/45 (31 %), and very low in 11/45
(24 %). TB treatment had already been initiated for 56 % (25/45) for a
median of 3 days (IQR, 1–10) at the time of sample collection (Table 1).
Of the 340 swabs collected from 41 prison cells with active TB patients,
Mtb DNA was detected in 55/340 (16 %) of ES swabs. We did not detect any
Mtb DNA (0/49) in swabs collected from non-TB lab and residential apart-
ments which served as negative controls. Mtb DNA positivity on custom
TaqMan assays was 6/70 (9 %) which was relatively higher on GeneXpert
MTB/RIF Ultra 49/270 (18 %). Among those positive on GeneXpert, the
majority were trace (37/49; 76 %) and remainder were low (5/49; 10 %)
or very low (7/49; 14 %). Mtb DNA was detected on various surfaces, and
positivity did not significantly differ across surface location or type: cloth-
ing, 25 % (2/8); bedding, 23 % (16/71); bathroom surfaces, 17 % (8/48);
personal utensils, 14 % (6/42); other surfaces in the cell, 13 % (23/171)
(Fig. 1A).
Of the 41 cells occupied by active TB patients, Mtb was detected in at
least one sample in 13/16 (81 %) of the cells housing TB patients with
high or medium Xpert sputum bacillary load and in 8/25 (32 %) of the
cells occupied by individuals with low or very low sputum Mtb load (p =
0.003; Fig. 1B). On a per-sample basis, 27 % of samples (44/161) were pos-
itive from cells with medium or high sputum Xpert cases and 6 % of samples
(11/179) were positive from cells with low or very low sputum Xpert. Mtb
4
Science of the Total Environment 843 (2022) 156970
Drug resistance detected Mutation site
No NA
Yes rpoB Ser450Leu
Yes rpoB Ser450Leu, katG 799_869del
Yes rpoB Ser450Leu
No NA
No NA
No NA
No NA
No katG Thr394Ala
No NA
detection on ES swabs was positively correlated with sputum Xpert bacil-
lary load (p < 0.001). To investigate whether the time at which cell was
swabbed has any impact on ES swab positivity, we ran logistic regression
model for positivity at the cell level as a function of duration on treatment.
We did not observe any significant association between cell positivity and
days since treatment initiation (p = 0.565).
3.2. Hybrid capture assays and whole genome sequencing
The seven captured ES swab samples had a median genomic coverage of
140× (range 23 to 209) (Supplementary Fig. 2). A mean of 98 % of the Mtb
genome had a depth of at least 10×; 65 % had a depth of at least 100×.
The median efficiency of our hybrid capture assay, which we defined
as the percentage of total sequenced reads that taxonomically assigned
to the Mycobacterium genus and were not assigned to a nontuberculous
Mycobacterium species was 92 % (range 65 % to 98 %) (Fig. 2).
3.3. Phylogenetic analysis
Mtb sequence data from captured ES swabs allowed us to assign taxon-
omy and investigate phylogenetic relatedness between samples. All seven
captured ES swab samples were assigned to lineage 4, the predominant lin-
eage in the state, and nested within previously sampled genomic diversity
from the state (Walter et al., 2022) (Fig. 3).
The three ES swabs from cell 1 were closely related (pairwise distance:
0–2 SNPs). In all three, we found evidence that both sub-lineages 4.8 and
4.1.2.1 were present; while the culture from that cell was assigned to line-
age 4.3.4.2 (Table 3). We predicted that consensus sequences generated
from environmental samples with two distinct sub-lineages could be chime-
ric, thus overestimating the genetic distance between these consensus
sequences and the matched culture. To test this, we excluded heterozygous
sites (Methods) from the environmental sample consensus sequences.
Revised consensus sequences from the three ES swab samples were identi-
cal to one another and were 23 SNPs distant from the matched cultured
isolate in cell 1 (85 SNPs distant before excluding heterozygous sites).
The three captured ES swab samples and one cultured sample from Cell 2
were all assigned to sub-lineage 4.1.2.1 and were closely related, with
consensus sequences 0–3 SNPs distant (Table 3). When excluding heterozy-
gous sites (Methods), consensus sequences were 0–2 SNPs distant.
Both sub-lineages 4.1.2.1 and 4.1.1.3 were present in the single
captured ES swab sample from cell 3 and the matched cultured sample
was assigned to sub-lineage 4.1.2.1 (Table 3). Again, we predicted that
the presence of multiple strains in the could lead to an over-estimate of
genetic distances between samples. We found that the environmental sam-
ples were genetically distant from the cultured isolate from the same cell
(pairwise distance: 55 SNPs; when excluding heterozygous sites: 23 SNPs).
3.4. Antibiotic resistance prediction
We predicted antimicrobial resistance profiles using a database of con-
firmed mutations associated with resistance phenotypes (Bradley et al.,
2015b). All three of the captured ES swab samples from cell 1 carried the
R. Verma et al.
(A)
Proportion of swabs with Mtb detected
0.6
0.4
0.2
0.0
n= n=
8 71
(B)
Proportion of cells with Mtb detected
0.75
0.50
0.25
0.00
High/Medium
Sputum GeneXpert grading
Fig. 1. (A) Mtb PCR positivity by surface type in prison cells. The samples were divided into five different categories- clothing (towel, shirt, trousers); bedding (bedsheets,
pillow covers, blanket and mattress); bathroom (sink, faucet, shower handle); personal utensils (cup, bowl, spoon and cigarette lighter); other surfaces in the cell (floor,
wall, window, ceiling). (B) Proportion of cells with positive Mtb PCR on ES swab. The cells were classified into two groups (high/medium and low/very low) based on
sputum Xpert bacillary load of the active TB patient present in the cell at the time of swab collection. The cell was counted as positive if Mtb DNA was detected in at least
one swab on GeneXpert MTB/RIF Ultra or RD-9 TaqMan assay.
same rifampicin resistance conferring mutation (rpoB Ser450Leu). All three
of these environmental samples were heteroresistant: both the susceptible
and resistant alleles were present at moderate coverage depth, possibly
on the distinct sub-lineages identified. 71 % (50/70) of reads corresponded
to the resistance-conferring mutation for the bed & clothing sample, 63 %
(85/134) for the utensils sample, and 60 % (47/78) from the other surfaces
sample. Additionally, a 70 base pairs deletion (799–869) in the katG gene
associated with isoniazid resistance, was present in 18 % of reads in the
cell 1 utensils sample. It is likely that the rpoB and katG mutations, sup-
ported by 63 % and 18 % of reads respectively, were on different genomic
backgrounds and do not reflect the presence of multi-drug resistant Mtb.
The corresponding culture sample from a patient in cell 1 was drug sensi-
tive. The remaining captured ES swab samples and corresponding cultures
from the two other cells contained no known drug-resistance conferring
mutations.
4. Discussion
In this study, we report the development and testing of a novel sampling
approach based on detecting Mtb DNA on environment surfaces where ac-
tive TB patients were present. We found that Mtb DNA was detectable in
most environments where individuals with active TB had recently resided,
and that detection was positively correlated with sputum bacillary load. By
adapting our sampling method for GeneXpert, which is widely available in
Science of the Total Environment 843 (2022) 156970
Sampling surfaces
Clothing
Bedding
Bathroom
Personal Utensils
Other surfaces
in the cell
n= n= n=
48 42 17
1
Low/Very low
tuberculosis-endemic countries, this approach could be utilized in resource-
constrained settings with minimal training or equipment. We further found
that we could sequence Mtb genomes directly from environmental samples
by using an RNA-bait hybrid capture approach, achieving sufficient cover-
age for lineage identification, phylogenetic analyses, and variant calling for
resistance-associated mutations. Taken together, these findings suggest that
environmental sampling could be a feasible approach for generating new
dimensions of data for studying tuberculosis transmission.
Several historical studies in late 19th and early 20th century demon-
strated the detection of Mtb in natural and built environments. In 1888,
Cornet collected dust from TB medical wards, residential apartments, and
asylums with active TB patients. The dust was subcutaneously inoculated
into guinea pigs. Among those inoculated with dust from TB sites, 16.4 %
developed TB, while none of the guinea pigs in negative control group
developed TB (Cornet, 1889). Later, in 1920, Rogers conducted similar
experiments by treating dust with 2 % sodium hydroxide solution subse-
quently inoculating the guinea pigs. In a series of experiments, seven out
of ten guinea pigs were diagnosed with disseminated TB (Rogers, 1920).
While these findings provide compelling evidence that Mtb may be detect-
able in the environment, most of these studies were performed in an era
when identification of Mtb at molecular level was not available. This is
particularly important in the context of environment samples, where slow
growing Mtb can be outgrown by other mycobacteria, which may also
share similar clinical or phenotypic features. In 2015, Velayati et al.,
5
R. Verma et al. Science of the Total Environment 843 (2022) 156970

Hyrbid capture-enriched genomes Non-enriched genomes

1.00

0.75
% Reads assigned

Taxonomic classification
Mycobacterium tuberculosis
Other mycobacterium genus
0.50
Nontuberculous mycobacteria
Unclassified
Other

0.25

0.00

Cell 3 Sputum culture

Cell 1 Sputum culture

Cell 2 Sputum culture

Cell 1 Utensils

Cell 2 Utensils

clothing
Cell 2 Bed &
surfaces
Cell 1 Other

Cell 3 Utensils
Cell 2 Lighter
clothing
Cell 1 Bed &

Fig. 2. Proportions of reads taxonomically assigned to the Mycobacterium tuberculosis species complex, the Mycobacterium genus (excluding the Mtb species complex or
nontuberculous Mycobacteria), nontuberculous Mycobacteria, unclassified, or mapping to other genera. Facets indicate the type of sample source: hybrid capture enriched
genomes from environment swabs and non-enriched genomes from sputum culture. Taxonomic classification was performed on trimmed reads using Kraken2.

attempted to isolate Mtb from 1500 randomly collected soil and water contrast, can be performed in seconds using low-cost disposable swabs. It
samples from three counties in Tehran (Velayati et al., 2015). The samples may be more sensitive, by detecting cumulative environmental contamina-
were inoculated on Löwenstein–Jensen culture media and monitored for tion, but does not provide the temporal resolution for recent presence of an
growth for several weeks. Mtb growth was observed in 1 % soil and 10 % infectious individual as does air sampling. Surface sampling may therefore
water samples. DNA isolated from positive cultures was used to perform be more useful for identifying high risk locations than for identifying the
spoligotyping and mycobacterial interspersed repetitive units-variable presence of infectious individuals.
number of tandem repeats (MIRU-VNTR) analysis. Typing of environmen- We detected Mtb DNA on multiple types of surfaces and locations,
tal and clinical isolates revealed partial overlap in Mtb diversity present in including vertical (walls) and horizontal ones (e.g. floors, bedding). While
the area (Velayati et al., 2015). These findings provided the first molecular the study was not designed to provide comparative information about loca-
evidence of detecting Mtb in environment. However, the screening strategy tions within rooms, we did not observe any systematic trends in positivity
employed was expensive, time consuming, labor intensive, and required by location or material type. Given that Mtb travels in aerosolized droplet
culturing hundreds of samples. Additionally, spoligotyping and MIRU- nuclei and can disseminate throughout rooms, it is likely to deposit on sur-
VNTR do not provide information on drug resistance and may lack suffi- faces throughout rooms. Based on our experience collecting and analyzing
cient data required to study transmission networks. Our novel sampling samples, we speculate that surfaces that are less frequently cleaned (e.g.
method allowed detection of Mtb on surfaces by direct swab sampling. By elevated or hard-to-reach surfaces) might be more likely to contain Mtb
pooling samples, we further sequenced Mtb from environment samples for DNA. Additionally, personal utensils which frequently come in close
lineage identification, phylogenetic analysis, and variant calling. To the proximity with the mouth of an infectious individual may also have higher
best of our knowledge, this is the first study to demonstrate the molecular chances of Mtb DNA detection if not cleaned thoroughly. Interestingly,
detection of Mtb in environment using targeted qPCR assays and whole we detected Mtb DNA on cigarette lighters from two cells analyzed on
genome sequencing. GeneXpert MTB/RIF Ultra. These lighters were re-swabbed after one
Previous studies have demonstrated that it is possible to detect Mtb from week for hybrid capture assays, and we detected Mtb DNA on one of the
air samples in high-risk settings such as hospitals, clinics, and schools in TB lighters which matched with the Mtb genome from paired sputum culture.
endemic settings (Bunyasi et al., 2022; Rufino de Sousa et al., 2020b; Additionally, a key variable which likely affects the recovery of Mtb DNA
Mastorides et al., 1999). The utility of air sampling is that it can be used from surfaces is how frequently they are cleaned. Cells from the study
to directly characterize airborne exposure risk. However, it requires that prisons were regularly cleaned; by contrast, tuberculosis isolation rooms
the infectious individual have been present in the sampling location within at the hospital only undergo light cleaning while occupied, followed by a
minutes of the sampling, which limits its sensitivity in communal settings. terminal decontamination when the patient leaves. It's possible that this
Additionally, air sampling requires more sophisticated equipment and (along with the high sputum smear grade) partially explains the higher
sampling that is conducted over a period of hours. Surface sampling, by recovery rate of Mtb from TB isolation rooms (supplementary material).

6
R. Verma et al.
ll 3
Ce
0.001
l1
el
C
Fig. 3. Maximum likelihood tree of 438 Mtb isolates from Mato Grosso do Sul, Brazil. The hybrid captured environmental and culture samples from patients within corre-
sponding cells nest within circulating Mtb diversity sampled through active and passive surveillance in the state. For the paired environmental and culture samples, tip
point colors and labels indicate cell and tip shapes indicate sample type. Phylogenetic branches are colored by assigned sublineage. Branch lengths are in units of substitution
per site.
Using RNA hybrid capture assays, we were able to enrich and fully se-
quence Mtb genomes from ES swabs, which generated sufficient coverage
for lineage identification and high-resolution variant calling. This is the
first use of hybrid capture for enriching Mtb from non-sputum samples
and demonstrates the potential for environmental sampling to inform
our understanding of circulating Mtb diversity, including that of drug-
resistance associated genotypes. ES swabs from two cells showed evidence
of mixed infection and did not match with the paired sputum culture
suggesting detection of an undiagnosed case or DNA from individual occu-
pied the cell previously. We also detected rpoB (S450L) and 70 base pairs
deletion in katG mutations conferring rifampin and isoniazid resistance
respectively. However, no patient-derived clinical isolate containing rifampi-
cin resistance was identified in that prison at the time of swab collection,
despite a concomitant mass screening campaign in which >90 % of prison
residents participated. One possible explanation to these unexpected find-
ings is frequent movement of incarcerated individuals between prisons
cells. This observation is particularly important in case of drug resistant TB,
where the infected individuals who usually share a cell with several healthy
individuals continue to amplify the transmission as they move from one cell
to another. This concerning finding suggests that ES sampling might provide
information on Mtb strains which were missed through clinical screening.
Our findings of Mtb DNA presence on environmental surfaces should
not be interpreted to imply that this represents an important route of trans-
mission. First, we used molecular assays that detect Mtb DNA and do not
distinguish between viable and dead organisms. While historical studies
7
Science of the Total Environment 843 (2022) 156970
Sample type
Bed & clothing
Sputum culture
Lighter
Other Surfaces
ll 2
C e ll 2
Ce
2
Cell
Cell
Cell 3
Cell 1
Cell 1 Cell 2
Cell 3
Lineage
4.1
4.3
4.4
4.7
4.8
4.9
found that Mtb may indeed survive for months in various environments,
there is little if any compelling data that transmission may occur from the
environment. The most interesting historical data concerning this possibil-
ity were from 100-year old studies showing that guinea pigs exposed to
reaersolized dust from sweeping the floor of tuberculosis wards were
infected, but there are no human data supporting infection through
reaerosolization of Mtb from surfaces (Cornet, 1889; Rogers, 1920). The
scientific understanding remains that infection is acquired predominantly
directly from human to human by exhaled aerosols. We believe the molec-
ular detection of Mtb in the environment can be most useful for understand-
ing where infection TB cases had been present, and perhaps as a means of
quantifying their infectiousness, rather than investigating the environment
as a route for transmission.
The results of this study should be interpreted with the context of sev-
eral limitations. First, we studied environments (hospital rooms, prisons)
in which individuals with TB are present for prolonged periods of time,
and positivity in environments where TB patients are present for shorter
periods requires further investigation. Future studies should evaluate
communal environments such as public transit, churches, schools, shops,
bars and other places where individuals congregate. Second, we could
only sequence Mtb captured on ES swabs from a small subset of prison
cells which had high ES swab positivity, and our findings comparing the
genomic data from environmental samples and clinical isolates were incon-
clusive. For example, rpoB mutations conferring rifampicin resistance were
present in three environmental swabs from one cell, yet the isolate from
R. Verma et al.
sputum culture did not contain rpoB mutations, and strains were genetically
distant, suggesting this was not explained by heteroresistance. We consider
our findings to demonstrate proof-of-concept that Mtb genome sequences
can be recovered from environmental samples, but their significance and
relation to infectious sources will require further studies with larger sample
sizes. In particular, the question of whether our environmental swab find-
ings overall reflect recent infectious cases or accumulation of Mtb DNA in
the environment needs further investigation. Lastly, our study lacks data
on Mtb culture from environment samples. We attempted to culture Mtb
from 75 swabs collected from positive prison cells (data not shown). How-
ever, none of the swab cultures were positive for Mtb after several weeks of
incubation. This could be due to differentially culturable organisms or
in vitro competition with other environmental organisms.
5. Conclusions
In summary, we found that Mtb DNA is frequently detectable in environ-
ments recently occupied by individuals with active TB. We demonstrated
that environmental sampling for Mtb can be performed using a simple
swabbing procedure and assayed on an automated molecular platform
widely used in low- and middle-income countries. We further found that
complete Mtb genome sequences can be recovered from environmental
samples using an RNA-bait hybrid capture approach, with sufficient depth
to allow variant calling and phylogenetic analysis. Our findings highlight
the potential for harnessing information derived from environmental sam-
pling in studying TB transmission.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2022.156970.
Data availability
Data supporting the findings of this manuscript are available in the
Supplementary Information files or from the corresponding author upon
request. Our bioinformatic pipeline is available at https://github.com/
ksw9/mtb_pipeline.
CRediT authorship contribution statement
Renu Verma: Study design; method development, validation, and data
collection; Formal analysis, Visualization; Writing – original draft & editing.
Flora Martinez Figueira Moreira: Sample collection; data collection;
Writing – review & editing. Agne Oliveira do Prado Morais: Sample
collection; Writing – review & editing. Katharine S. Walter: Writing –
original draft & editing, data analysis and visualization. Paulo César
Pereira dos Santos: Sample collection; data collection; Writing – review
& editing. Eugene Kim: Data collection; Writing – review & editing.
Thiego Ramon Soares: Sample collection; Writing – review & editing.
Rafaele Carla Pivetta de Araujo: Sample collection; Writing – review &
editing. Bruna Oliveira da Silva: Sample collection; Writing – review &
editing. Andrea da Silva Santos: Sample collection; Writing – review &
editing. Julio Croda: Study design; Supervision; Formal analysis; Writing –
review & editing Jason R. Andrews: Conceptualization; Supervision;
Study design; Writing – original draft & editing; Funding acquisition;
Formal analysis; sample collection.
Declaration of competing interest
The authors declare the following financial interests/personal relation-
ships which may be considered as potential competing interests: Jason R.
Andrews reports financial support was provided by National Institutes of
Health.
Acknowledgements
We thank the prison staff for their support and incarcerated individuals
from prisons Penitenciária Estadual de Dourados (PED) and Estabelecimento
8
Science of the Total Environment 843 (2022) 156970
Penal Jair Ferreira de Carvalho (EPJFC), Brazil for collecting ES swabs from
their cells. We also thank Leonardo Martinez from Boston University for his
support.
Funding
This study was supported by NIH DP2 AI131082 and NIH AI130058.
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