The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20؋ depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.T he global incidence of multidrug-resistant (MDR), extensively drug-resistant (XDR), and totally drug-resistant tuberculosis (TB) has risen over the last decade (1), making it increasingly important to rapidly and accurately detect resistance. The gold standard for antimicrobial resistance testing relies on bacterial culture, which can take upwards of several weeks for Mycobacterium tuberculosis. Molecular tests, such as the Xpert (MTB/RIF) and line probe assays, which can be used directly on sputum have improved identification of MDR M. tuberculosis but are able to identify only limited numbers of specific resistance mutations (2, 3).Whole-genome sequencing (WGS) of bacterial genomes allows simultaneous identification of all known resistance mutations as well as markers with which transmission can be monitored (4). WGS of M. tuberculosis provides resolution superior to that of other current methods such as spoligotyping and mycobacterial interspersed repetitive-unit-variable-number tandemrepeat (MIRU-VNTR) analysis for strain genotyping (5), and its usefulness in defining outbreaks has been demonstrated previously (6-9). Currently, however, WGS of M. tuberculosis requires prior bacterial enrichment by culturing and most outbreak studies have therefore been retrospective (6-8). Recently, WGS of M. tuberculosis has been achieved ...

Background: Immunological ex vivo assays to diagnose tuberculosis (TB) have great potential but have largely been blood-based and poorly evaluated in active TB. Lung sampling enables combined microbiological and immunological testing and uses higher frequency antigenspecific responses than in blood. Methods: A prospective evaluation was undertaken of a flow cytometric assay measuring the percentage of interferon-c synthetic CD4+ lymphocytes following stimulation with purified protein derivative of Mycobacterium tuberculosis (PPD) in bronchoalveolar lavage fluid from 250 sputum smear-negative individuals with possible TB. A positive assay was defined as .1.5%. Results: Of those who underwent lavage and were diagnosed with active TB, 95% (106/111) had a positive immunoassay (95% CI 89% to 98%). In 139 individuals deemed not to have active TB, 105 (76%) were immunoassay negative (95% CI 68% to 82%). Of the remaining 24% (34 cases) with a positive immunoassay, a substantial proportion had evidence of untreated TB; in two of these active TB was subsequently diagnosed. Assay performance was unaffected by HIV status, disease site or BCG vaccination. In culture-positive pulmonary cases, response to PPD was more sensitive than nucleic acid amplification testing (94% vs 73%). The use of early secretory antigen target-6 (ESAT-6) responses in 71 subjects was no better than PPD, and 19% of those with culture-confirmed TB and a positive PPD immunoassay had no detectable response to ESAT-6. Conclusions: These findings suggest that lung-orientated immunological investigation is a potentially powerful tool in diagnosing individuals with sputum smear-negative active TB, regardless of HIV serostatus.

The Ebola virus disease (EVD) outbreak in West Africa was unprecedented in scale and location. Limited access to both diagnostic and supportive pathology assays in both resource-rich and resource-limited settings had a detrimental effect on the identification and isolation of cases as well as individual patient management. Limited access to such assays in resource-rich settings resulted in delays in differentiating EVD from other illnesses in returning travellers, in turn utilising valuable resources until a diagnosis could be made. This had a much greater impact in West Africa, where it contributed to the initial failure to contain the outbreak. This review explores diagnostic assays of use in EVD in both resource-rich and resource-limited settings, including their respective limitations, and some novel assays and approaches that may be of use in future outbreaks.