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BACKGROUND: Whole genome sequencing (WGS) is rarely used for drug-resistance testing of Mycobacterium tuberculosis in high-endemic settings. We present the first study from Indonesia, which has the second highest tuberculosis (TB) burden worldwide, with less than 50% of drug-resistant cases currently detected. METHODS: We applied WGS in strains from 322 adult HIV-negative TB patients. Phenotypic DST was done for a portion of patients. RESULTS: Fifty-one isolates (15.8%) harboured drug resistance mutations, including 42 among 322 patients (13.0%) with no prior TB treatment. Eight (2.5%) isolates were multidrug-resistant (MDR), one was extensively drug-resistant (XDR). Most mutations were found in katG (n=18), pncA (n=18), rpoB (n=10), fabG1 (n=9) and embB (n=9). The agreement of WGS-based resistance and phenotypic drug susceptibility testing (DST) to first-line drugs was high for isoniazid, rifampicin, and streptomycin, and less for ethambutol. Drug resistance was more common in Indo-Oceanic lineage strains (37.5%) than Euro-American (18.2%) and East-Asian lineage strains (10.3%; p=0.044), but combinations of multiple mutations were most common among East-Asian lineage strains (p=0.054). CONCLUSIONS: Our data support the potential use of WGS for more rapid and comprehensive prediction ofDR-TB in Indonesia. Future studies should address potential barriers in implementing WGS, the distribution of specific resistance mutations, and associations of particular mutations with endemic M. tuberculosis lineages in Indonesia.

Objectives Whole-genome sequencing (WGS) is rarely used for drug resistance testing of Mycobacterium tuberculosis in high-endemic settings. Here we present the first study from Indonesia, which has the third highest tuberculosis (TB) burden worldwide, with Methods WGS was applied for strains from 322 human immunodeficiency virus (HIV)-negative adult TB patients. Phenotypic drug susceptibility testing (DST) was performed for a proportion of the patients. Results Using WGS, mutations associated with drug resistance to any TB drug were identified in 51 (15.8%) of the 322 patients, including 42 patients (13.0%) with no prior TB treatment (primary resistance). Eight isolates (2.5%) were multidrug-resistant (MDR) and one was extensively drug-resistant (XDR). Most mutations were found in katG (n = 18), pncA (n = 18), rpoB (n = 10), fabG1 (n = 9) and embB (n = 9). Agreement of WGS-based resistance and phenotypic DST to first-line drugs was high for isoniazid and rifampicin but was lower for ethambutol and streptomycin. Drug resistance was more common in Indo-Oceanic lineage strains (37.5%) compared with Euro-American (18.2%) and East-Asian lineage strains (10.3%) (P = 0.044), but combinations of multiple mutations were most common among East-Asian lineage strains (P = 0.054). Conclusions These data support the potential use of WGS for more rapid and comprehensive prediction of drug-resistant TB in Indonesia. Future studies should address potential barriers to implementing WGS, the distribution of specific resistance mutations, and the association of particular mutations with endemic M. tuberculosis lineages in Indonesia.

Supplementary methods and Figure to 'Diabetes is associated with genotypically drug-resistant tuberculosis'.

Supplementary methods and Figure to 'Diabetes is associated with genotypically drug-resistant tuberculosis'.

Protein prediction for genomic sites associated with the TB disease phenotype. NA: No homologs of Rv0192 were found therefore protein prediction was not possible. # I-mutant predicts free energy changes of protein stability upon a point mutation under different conditions. & PolyPhen predicts the possible impact of an amino acid substitution on the structure and function of a human protein using straightforward physical and comparative considerations. ^ TargetP predicts the subcellular location of proteins based on the predicted presence of any N-terminal signal peptides. * TMHMM predicts transmembrane helices in proteins. (DOCX 57Â kb)

PE / PPE genes and drug resistance genes excluded for the phylogeny construction. Listed are the genes that were excluded from the multiple alignment used to create the phylogenetic tree. (DOCX 158Â kb)

Tuberculous meningitis (TBM) is the most severe and disabling form of tuberculosis (TB), accounting for around 1-5% of the global TB caseload, with mortality of approximately 20% in children and up to 60% in persons co-infected with human immunodeficiency virus even in those treated. Relatively few centres of excellence in TBM research exist and the field would therefore benefit from greater co-ordination, advocacy, collaboration and early data sharing. To this end, in 2009, 2015 and 2019 we convened the TBM International Research Consortium, bringing together approximately 50 researchers from five continents. The most recent meeting took place on 1 st and 2 nd March 2019 in Lucknow, India. During the meeting, researchers and clinicians presented updates in their areas of expertise, and additionally presented on the knowledge gaps and research priorities in that field. Discussion during the meeting was followed by the development, by a core writing group, of a synthesis of knowledge gaps and research priorities within seven domains, namely epidemiology, pathogenesis, diagnosis, antimicrobial therapy, host-directed therapy, critical care and implementation science. These were circulated to the whole consortium for written input and feedback. Further cycles of discussion between the writing group took place to arrive at a consensus series of priorities. This article summarises the consensus reached by the consortium concerning the unmet needs and priorities for future research for this neglected and often fatal disease.

Ancestral reconstruction of SNP 261869TC in Rv0218. Listed are the internal nodes and leaves where the SNP in Rv0218 occurred according to the ancestral reconstruction of the SNP. (DOCX 72Â kb)

Description of baseline characteristics for PTB and TBM patients. IQR, interquartile range; SD, standard deviation. Data were missing for history of TB treatment (TBM, n = 7; PTB, n = 1); ethnicity (TBM, n = 58; PTB = 2). (DOCX 58 kb)

Read alignment demonstrating the absence of nanK. Displayed is the alignment of the raw sequencing reads against the H37Rv nanK gene. No reads are mapping to this gene, showing that it is absent in the sequenced genome. (DOCX 54Â kb)

Tuberculous meningitis (TBM) is the most severe and disabling form of tuberculosis (TB), accounting for around 1-5% of the global TB caseload, with mortality of approximately 20% in children and up to 60% in persons co-infected with human immunodeficiency virus even in those treated. Relatively few centres of excellence in TBM research exist and the field would therefore benefit from greater co-ordination, advocacy, collaboration and early data sharing. To this end, in 2009, 2015 and 2019 we convened the TBM International Research Consortium, bringing together approximately 50 researchers from five continents. The most recent meeting took place on 1 st and 2 nd March 2019 in Lucknow, India. During the meeting, researchers and clinicians presented updates in their areas of expertise, and additionally presented on the knowledge gaps and research priorities in that field. Discussion during the meeting was followed by the development, by a core writing group, of a synthesis of knowledge gaps and research priorities within seven domains, namely epidemiology, pathogenesis, diagnosis, antimicrobial therapy, host-directed therapy, critical care and implementation science. These were circulated to the whole consortium for written input and feedback. Further cycles of discussion between the writing group took place to arrive at a consensus series of priorities. This article summarises the consensus reached by the consortium concerning the unmet needs and priorities for future research for this neglected and often fatal disease.

Description of baseline characteristics for PTB and TBM patients. IQR, interquartile range; SD, standard deviation. Data were missing for history of TB treatment (TBM, n = 7; PTB, n = 1); ethnicity (TBM, n = 58; PTB = 2). (DOCX 58 kb)

Protein prediction for genomic sites associated with the TB disease phenotype. NA: No homologs of Rv0192 were found therefore protein prediction was not possible. # I-mutant predicts free energy changes of protein stability upon a point mutation under different conditions. & PolyPhen predicts the possible impact of an amino acid substitution on the structure and function of a human protein using straightforward physical and comparative considerations. ^ TargetP predicts the subcellular location of proteins based on the predicted presence of any N-terminal signal peptides. * TMHMM predicts transmembrane helices in proteins. (DOCX 57Â kb)

Diagram demonstrating breseq calling a SNP in the PE-PGRS1 gene. Displayed are 60 Illumina sequencing reads mapping to the H37Rv reference genome (shown at the top and bottom). Visual inspection of the SNP confirms that it does not occur in a region containing uniformly lower base quality scores. (DOCX 1204Â kb)

Read alignment demonstrating the absence of nanK. Displayed is the alignment of the raw sequencing reads against the H37Rv nanK gene. No reads are mapping to this gene, showing that it is absent in the sequenced genome. (DOCX 54Â kb)

Ancestral reconstruction of SNP 261869TC in Rv0218. Listed are the internal nodes and leaves where the SNP in Rv0218 occurred according to the ancestral reconstruction of the SNP. (DOCX 72Â kb)

P-values of association between SNPs and TB disease phenotype in the discovery and validation sets. Scatterplot showing the p-values of the SNPs found in the discovery and the validation set. P-values in the discovery set are shown on the x-axis; p-values in the validation set are shown on the y-axis. Gene names are shown for SNPs significant in both the discovery and validation set. (DOCX 82Â kb)

Phylogenetic tree of 322Â M. tuberculosis strains isolated from TBM and PTB patients. The highlighted branches indicate the 108 strains in 47 terminal branch sets, together comprising the discovery set. The purple stars indicate the origin of the SNP in Rv0218 according to the ancestral reconstruction. The nucleotide for SNP position 261,869 is indicated next to the leaf labels. (PDF 60Â kb)

Phylogenetic tree of 322Â M. tuberculosis strains isolated from TBM and PTB patients. The highlighted branches indicate the 108 strains in 47 terminal branch sets, together comprising the discovery set. The purple stars indicate the origin of the SNP in Rv0218 according to the ancestral reconstruction. The nucleotide for SNP position 261,869 is indicated next to the leaf labels. (PDF 60Â kb)

PE / PPE genes and drug resistance genes excluded for the phylogeny construction. Listed are the genes that were excluded from the multiple alignment used to create the phylogenetic tree. (DOCX 158Â kb)

Description of sequencing quality control parameters and statistics. Displayed are different measures of sequencing quality, used for the sequencing quality control check. (DOCX 208Â kb)

P-values of association between genes and TB disease phenotype in the discovery and validation sets. Scatterplot showing the p-values of the genes found in the discovery and the validation set. P-values in the discovery set are shown on the x-axis; p-values in the validation set are shown on the y-axis. Names are shown for genes significant in both the discovery and validation set. (DOCX 66Â kb)

Diagram demonstrating breseq calling a SNP in the PE-PGRS1 gene. Displayed are 60 Illumina sequencing reads mapping to the H37Rv reference genome (shown at the top and bottom). Visual inspection of the SNP confirms that it does not occur in a region containing uniformly lower base quality scores. (DOCX 1204Â kb)