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Successful transmission of tuberculosis depends on the interplay of human behavior, host immune responses, and Mycobacterium tuberculosis virulence factors. Previous studies have been focused on identifying host risk factors associated with increased transmission, but the contribution of specific genetic variations in mycobacterial strains themselves are still unknown., This study was funded by the Portuguese Foundation for Science and Technology (FCT) (SFRH/BD/33902/2009 [H.N.-G.]).

Item does not contain fulltext RATIONALE: The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs. OBJECTIVES: To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance. METHODS: We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool. MEASUREMENTS AND MAIN RESULTS: The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci. CONCLUSIONS: These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs.

Rationale: Mutations in the cystic fibrosis (CF) transmembrane conductance regulator affect the innate epithelial immune function of the lung, resulting in exaggerated and ineffective airway inflammation that fails to eradicate pathogenic fungi. The appreciation of whether such fungi are primarily responsible for or a consequence of ineffective airway inflammation is important for future therapeutics development.Objectives: To characterize the impact of the tryptophan/kynurenine pathway on pathogenic airway inflammation preventing effective fungal clearance in CF.Methods: We studied the expression of indoleamine 2,3-dioxygenase (IDO), the first enzyme in the kynurenine pathway of tryptophan degradation, in human and murine CF, the impact of IDO on lung inflammation and immunity in murine CF, and the potential role of tryptophan catabolism in pathogenesis and therapy of fungus-associated lung inflammation.Measurements and Main Results: IDO was defective in murine and human CF. Genetic and transcriptional regulatory mechanisms contributed to dysfunctional IDO activity that, in turn, correlated with imbalanced Th17/Treg-cell responses to Aspergillus fumigatus in murine CF. Treatments enhancing IDO function or preventing pathogenic Th17-cell activation restored protective immunity to the fungus and improved lung inflammation in murine CF.Conclusions: This study provides a link between tryptophan catabolism and lung immune homeostasis in murine CF, representing a proof-of-concept that targeting pathogenic inflammation via IDO-mimetic drugs may benefit patients with CF., Supported by a grant from the Italian Cystic Fibrosis Research Foundation (Research Project no. FFC#21/2010 to L. Romani) with the contribution of Francesca Guadagnin, Coca Cola Light Tribute to Fashion, and Delegazione FFC di Belluno; and the Specific Targeted Research Project "ALLFUN" (FP7-HEALTH-2009 contract no. 260338 to L. Romani). R.G.I. gratefully acknowledges a fellowship from the Italian Cystic Fibrosis Research Foundation. A. Carvalho and C. Cunha were financially supported by fellowships from the Fundacao para a Ciencia e Tecnologia, Portugal (contracts SFRH/BPD/46292/2008 and SFRH/BD/65962/2009, respectively).