Nontuberculous Mycobacteria

Sputum samples were decontaminated according to the sodium
hydroxide–N-acetyl-L-cysteine method.21 An aliquot was used for
microscopical examination of auramine-stained sputum smears, and the
remainder was used for parallel Löwenstein–Jensen
culture, automated mycobacterial culture, and MODS culture (see Fig. I in
the Supplementary Appendix, available with the full text of this article at
www.nejm.org). Löwenstein–Jensen culture
and automated mycobacterial culture with the use of the MBBacT system
(bioMérieux) were selected because they are reference methods
commonly used in developing and industrialized countries, respectively.
After inoculation of 250 μl of decontaminant,
Löwenstein–Jensen slants were incubated at
37°C and examined twice weekly from day 7 through day 60.21 MBBacT bottles were inoculated with
500 μl of decontaminant, and cultures were monitored
continuously for 42 days according to the recommendations of the
manufacturer.
The MODS assay was performed as described previously.6 (link),7 (link)
Briefly, broth cultures were prepared in 24-well tissue-culture plates
(Becton Dickinson), each containing 720 μl of decontaminant,
Middlebrook 7H9 broth (Becton Dickinson), oxalic acid, albumin, dextrose,
and catalase (OADC) (Becton Dickinson), and polymyxin, amphotericin B,
nalidixic acid, trimethoprim, and azlocillin (PANTA) (Becton Dickinson). For
each sample, 12 wells were used: in 4 control wells, no drug was added, and
each of the remaining 8 wells contained one of four drugs at one of two
concentrations tested. The cultures were examined under an inverted light
microscope at a magnification of 40× every day (except Saturday
and Sunday) from day 4 to day 15, on alternate days from day 16 to day 25,
and twice weekly from day 26 to day 40. To minimize cross-contamination and
occupational exposure, plates were permanently sealed inside plastic ziplock
bags after inoculation and were subsequently examined within the bag.
Positive cultures were identified by cord formation, characteristic of
M. tuberculosis growth, in liquid medium in drug-free
control wells, as described previously.6 (link),7 (link),22 (link) Nontuberculous mycobacteria were recognized by
their lack of cording or, for M. chelonae (which is the
only nontuberculous mycobacteria that does form cords), by rapid overgrowth
by day 5. Fungal or bacterial contamination was recognized by rapid
overgrowth and clouding in wells.
If contamination was detected, the original sample was cultured
again after being decontaminated once more. Spacer oligonucleotide typing
(spoligotyping), polymerase chain reaction with multiple primers,23 (link) or both were applied to all isolates
from each of the three types of cultures in order to confirm the presence of
M. tuberculosis.

Raw read data for 8,136 strains were downloaded from the SRA (see SRA accessions in Supplementary Table 1). Reads were mapped onto a reference strain of H37Rv (GenBank accession number CP003248.2) using BWA version 0.7.10^{68 (link)}. Variants were identified using Pilon version 1.11 as described ^{67 (link)}. The global M. tuberculosis lineage designations used in our analysis, as well as each strain’s spoligotype, were predicted using digital spoligotyping as in Cohen et al., 2015⁶ .
We eliminated 824 strains that did not pass our quality control filters: average sequencing depth of coverage >20X; fraction of long insertions <0.2; ambiguity rate <0.5 (to remove samples that were suspected to represent mixes of different genotypes); number of low coverage bases <250,000; and having a single match to one lineage in our lineage-prediction algorithm. We also eliminated strains for which Pilon failed three times. Of the remaining 7,312 samples, we removed 1,970 strains with no “country” metadata or description in a publication; 19 strains with substantial non-tuberculous mycobacteria contamination; as well as 13 additional duplicate patient samples. These filters resulted in a final set of 5,310 strains for analysis.
Emu⁶⁹ was run to canonicalize variants. We conducted phylogenetic analyses for the entire set of 5,310 strains, as well as for a subset corresponding to each lineage and each United Nations geographical subregion²³ with >30 strains (Supplementary Table 3). For each set, all sites with unambiguous single nucleotide polymorphisms (SNPs) in at least one strain were combined into a concatenated alignment. Ambiguous positions were treated as missing data. The concatenated alignment was then were used to generate a midpoint rooted phylogenetic tree using FastTree^{70 (link)} version 2.1.8.