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Background: Drug-susceptibility testing (DST) of Mycobacterium tuberculosis complex (MTBC) isolates by the Mycobacteria Growth Indicator Tube (MGIT) approach is the most widely applied reference standard. However, the use of WGS is increasing in many developed countries to detect resistance and predict susceptibility. We investigated the reliability of WGS in predicting drug susceptibility, and analysed the discrepancies between WGS and MGIT against the first-line drugs rifampicin, isoniazid, ethambutol and pyrazinamide., Methods: DST by MGIT and WGS was performed on MTBC isolates received in 2016/2017. Nine genes and/or their promotor regions were investigated for resistance-associated mutations: rpoB, katG, fabG1, ahpC, inhA, embA, embB, pncA and rpsA. Isolates that were discrepant in their MGIT/WGS results and a control group with concordant results were retested in the MGIT, at the critical concentration and a lower concentration, and incubated for up to 45 days after the control tube became positive in the MGIT., Results: In total, 1136 isolates were included, of which 1121 were routine MTBC isolates from the Netherlands. The negative predictive value of WGS was ≥99.3% for all four first-line antibiotics. The majority of discrepancies for isoniazid and ethambutol were explained by growth at the lower concentrations, and for rifampicin by prolonged incubation in the MGIT, both indicating low-level resistance., Conclusions: Applying WGS in a country like the Netherlands, with a low TB incidence and low prevalence of resistance, can reduce the need for phenotypic DST for ∼90% of isolates and accurately detect mutations associated with low-level resistance, often missed in conventional DST., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Introduction: Tuberculosis remains a global health problem and pharmacokinetic variability has been postulated as one of the causes of treatment failure and acquired drug resistance. New developments enable implementation of therapeutic drug monitoring, a strategy to evaluate drug exposure in order to tailor the dose to the individual patient, in tuberculosis treatment., Areas Covered: Literature on pharmacokinetics and pharmacodynamics of anti-tuberculosis drugs was explored to evaluate the effect of drug exposure in relation to drug susceptibility, toxicity and efficacy. New, down-sized strategies, like dried blood spot analysis and limited sampling strategies are reviewed. In addition, molecular resistance testing of Mycobacteria tuberculosis, combining a short turn-around time with relevant information on drug susceptibility of the causative pathogen was explored. Newly emerging host biomarkers provide information on the response to treatment., Expert Opinion: Therapeutic drug monitoring can minimize toxicity and increase efficacy of tuberculosis treatment and prevent the development of resistance. Dried blood spot analysis and limited sampling strategies, can be combined to provide us with a more patient friendly approach. Furthermore, rapid information on drug susceptibility by molecular testing, and information from host biomarkers on the bacteriological response, can be used to further optimize tuberculosis treatment.

Objectives: Fluoroquinolones are used in second-line treatment of tuberculosis (TB) and have a potential role in shortening TB treatment duration. The wide use of fluoroquinolones in the treatment of other infections, including respiratory tract infections in patients with (undiagnosed) active TB, could result in fluoroquinolone-resistant Mycobacterium tuberculosis. We determined the rate of fluoroquinolone resistance in M. tuberculosis isolates obtained from Tanzanian patients and linked this to previous fluoroquinolone exposure and mycobacterial resistance to rifampicin and isoniazid., Methods: A total of 291 M. tuberculosis isolates were obtained between April 2009 and June 2010 from patients with smear-positive pulmonary TB and tested for susceptibility to ciprofloxacin, moxifloxacin, rifampicin and isoniazid. Information on previous fluoroquinolone use was obtained by interviewing patients and checking their medical files., Results: Only 2 (0.7%) of the 291 M. tuberculosis isolates were resistant to ciprofloxacin; 1 of which was intermediately resistant to moxifloxacin as well. These two isolates were susceptible to rifampicin and isoniazid. Twenty-two (8%) of the 291 patients had a history of fluoroquinolone use (median: 7 days; interquartile range: 5-10 days). The patients from whom the fluoroquinolone-resistant M. tuberculosis isolates were obtained had no known history of previous fluoroquinolone use., Conclusions: Our findings indicate that the rate of fluoroquinolone-resistant M. tuberculosis in Tanzanian patients with TB is low and not related to previous, brief episodes of exposure to fluoroquinolones. The findings favour future application of fluoroquinolones in TB treatment regimens of shorter duration.

In general non-human primates are highly susceptible to infections with Mycobacterium tuberculosis which therefore presents an explosive health threat to colonies. To screen for M. tuberculosis infections in non-human primates, the skin test is routinely used. However, the reliability of this test in primates is debatable. The aim of this study was to compare relatively easy in vitro diagnostic tests for TB with the skin test for detection of a tuberculosis (TB) infection. Two in vitro assays, a whole blood interferon-gamma (WB IFN-gamma) assay and in vitro stimulation of isolated lymphocytes (PBMC IFN-gamma) were evaluated during both experimental TB infections in macaques as well as during an outbreak of TB in a macaque quarantine facility. The WB IFN-gamma assay was also evaluated on healthy old and new world monkeys. Our results show that both in vitro assays detected TB infection in macaques. All experimentally infected animals showed TB-specific responses in both assays. In contrast, several TB animals were not diagnosed TB positive using the skin test. In addition, during the outbreak in the quarantine facility one animal was not detected using the routinely used skin test, but it showed strong positive responses in the WB assay. In conclusion, the in vitro assays are a valuable tool for screening non-human primates for TB infection, especially because the assays cause relatively less stress for the animals compared to the skin test and give reproducible and reliable results.

Despite the widespread use of bacillus Calmette-Guérin vaccination, Mycobacterium tuberculosis infection remains globally the leading cause of death from a single infectious disease. The complicated and often protracted dynamics of infection and disease make clinical trials to test new tuberculosis vaccines extremely complex. Preclinical selection of only the most promising candidates is therefore essential. Because macaque monkeys develop a disease very similar to humans, they have potential to provide important information in addition to small animal models. To assess the relative merits of rhesus and cynomolgus monkeys as screens for tuberculosis vaccines, we compared the efficacy of bacillus Calmette-Guérin vaccination and the course of infection in both species. Unvaccinated rhesus and cynomolgus monkeys both developed progressive disease with high levels of C-reactive protein, M. tuberculosis-specific IgG, and extensive pathology including cavitation and caseous necrosis. Bacillus Calmette-Guérin vaccination protected cynomolgus almost completely toward the development of pathology, reflected in a striking 2-log reduction in viable bacteria in the lungs compared with nonvaccinated animals. Rhesus, on the other hand, were not protected efficiently by the bacillus Calmette-Guérin. The vaccinated animals developed substantial pathology and had negligible reductions of colony-forming units in the lungs. Comparative studies in these closely related species are likely to provide insight into mechanisms involved in protection against tuberculosis.