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Nontuberculous mycobacteria include 198 mycobacterial species. Among these, Mycobacteroides abscessus is a rapidly growing mycobacteria that causes lung and skin infections. M. abscessus lung infections are difficult to treat due to the high levels of resistance to several classes of antibiotics. The current treatment is based on combining at least two or three antibiotics. However, treatment outcomes remain very poor. The objective was to compare the in vitro activity of amikacin, tigecycline, imipenem, and clarithromycin, alone and in two different three-drug combinations (amikacin/tigecycline/imipenem and amikacin/tigecycline/clarithromycin) against seven M. abscessus subsp. abscessus clinical isolates using the time-kill assay. The two combinations showed greater activity than the antibiotics tested individually. Even though both combinations showed similar activity as well as no antagonistic activity, the combination including imipenem could not be an alternative treatment against M. abscessus subsp. abscessus lung infections caused by clarithromycin susceptible isolates. However, this combination could be considered against clarithromycin resistant isolates. Future studies are necessary to confirm this hypothesis This work was supported by the Ministerio de Economía y Competitividad, Instituto de Salud Carlos III, co-financed by the European Regional Development Fund (ERDF) ‘A way to achieve Europe’, the Spanish Ministry of Health (grant no. PI16/01047), Planes Nacionales de I+D+i 2008–2011/2013–2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, Spanish Network for Research in Infectious Diseases (REIPI) (RD16/ 0016/0010) co‐financed by European Development Regional Fund (ERDF) “A way to achieve Europe” and operative program Intelligent Growth 2014–2020. This study was also supported by grant 2017SGR0809 from the Departament d’Universitats, Recerca i Societat de la Informació de la Generalitat de Catalunya and by a grant from Fundació La Marató de TV3 (grant no. 201816-10). EP-B received a grant from the Universitat de Barcelona (UB), Ajut de Personal Investigador en Formació (APIF-UB)

The main objective of this study was to compare in vitro activities of a novel fluoroquinolone (FQ), UB-8902, with ofloxacin (OFX), levofloxacin (LFX), and moxifloxacin (MOX) against Mycobacterium ...

Rifampicin heteroresistance – where rifampicin-resistant and -susceptible tuberculosis (TB) bacilli co-exist – may result in failed standard TB treatment and potential spread of rifampicin-resistant strains. Detection of rifampicin heteroresistance in routine rapid diagnostic tests (RDTs) allows for patients to receive prompt and effective multidrug-resistant-TB treatment, and may improve rifampicin-resistant TB control.The limit of detection (LOD) of rifampicin heteroresistance for phenotypic drug susceptibility testing by the proportion method is 1%, yet is insufficiently documented for RDTs. We therefore aimed to determine, for the four RDTs (XpertMTB/RIF, XpertMTB/RIF Ultra, GenoTypeMTBDRplusv2.0, and GenoscholarNTM+MDRTBII), the LOD per probe and mutation, validated by colony-forming-unit-counting and targeted deep sequencing (Deeplex-MycTB).We selected one rifampicin-susceptible and four rifampicin-resistant strains, with mutation D435V, H445D, H445Y, and S450L respectively, mixed them in various proportions in triplicate, tested them with each RDT, and determined the LODs per mutation type. Deeplex-MycTB revealed concordant proportions of the minority resistant variants in the mixtures. The Deeplex-MycTB-validated-LODs ranged from 20-80% for XpertMTB/RIF, 20-70% for Xpert Ultra, 5-10% for GenoTypeMTBDRplusv2.0, and 1-10% for GenoscholarNTM+MTBII for the different mutations.Deeplex-MycTB, GenoTypeMTBDRplusv2.0, and GenoscholarNTM+MDRTBII, provide explicit information on rifampicin heteroresistance for the most frequently detected mutations. Classic Xpert and Ultra report rifampicin heteroresistance as rifampicin resistance, while Ultra may denote rifampicin heteroresistance through ‘mixed patterns’ of wild-type and mutant melt probe melt peak temperatures.Overall, our findings inform end-users that the threshold for reporting resistance in case of rifampicin heteroresistance is the highest for Classic Xpert and Ultra, to resolve phenotypic and genotypic discordant rifampicin-resistant TB results.

Tuberculosis (TB) remains a major threat to human health worldwide. The increasing incidence of non-tuberculous mycobacterial infections and particularly those produced by Mycobacterium avium has emphasized the need to develop new drugs. Additionally, high levels of natural drug resistance in non-tuberculous mycobacteria (NTM) and the emergence of multidrug-resistant (MDR) TB is of great concern. Antimicrobial peptides (AMPs) are antibiotics with broad-spectrum antimicrobial activity. The objective was to assess the activity of AMPs against Mycobacterium tuberculosis and M. avium clinical isolates. MICs were determined using microtitre plates and the resazurin assay. Mastoparan and melittin showed the greatest activity against M. tuberculosis, while indolicidin had the lowest MIC against M. avium. In conclusion, AMPs could be alternatives for the treatment of mycobacterial infections. Further investigation of AMPs' activity in combination and associated with conventional antibiotics and their loading into drug-delivery systems could lead to their use in clinical practice.

Rifampin heteroresistance—where rifampin-resistant and -susceptible tuberculosis (TB) bacilli coexist—may result in failed standard TB treatment and potential spread of rifampin-resistant strains. The detection of rifampin heteroresistance in routine rapid diagnostic tests (RDTs) allows for patients to receive prompt and effective multidrug-resistant-TB treatment and may improve rifampin-resistant TB control., Rifampin heteroresistance—where rifampin-resistant and -susceptible tuberculosis (TB) bacilli coexist—may result in failed standard TB treatment and potential spread of rifampin-resistant strains. The detection of rifampin heteroresistance in routine rapid diagnostic tests (RDTs) allows for patients to receive prompt and effective multidrug-resistant-TB treatment and may improve rifampin-resistant TB control. The limit of detection (LOD) of rifampin heteroresistance for phenotypic drug susceptibility testing by the proportion method is 1% and, yet, is insufficiently documented for RDTs. We, therefore, aimed to determine, for the four RDTs (XpertMTB/RIF, XpertMTB/RIF Ultra, GenoTypeMTBDRplusv2.0, and GenoscholarNTM+MDRTBII), the LOD per probe and mutation, validated by CFU counting and targeted deep sequencing (Deeplex-MycTB). We selected one rifampin-susceptible and four rifampin-resistant strains, with mutations D435V, H445D, H445Y, and S450L, respectively, mixed them in various proportions in triplicate, tested them with each RDT, and determined the LODs per mutation type. Deeplex-MycTB revealed concordant proportions of the minority resistant variants in the mixtures. The Deeplex-MycTB-validated LODs ranged from 20% to 80% for XpertMTB/RIF, 20% to 70% for Xpert Ultra, 5% to 10% for GenoTypeMTBDRplusv2.0, and 1% to 10% for GenoscholarNTM+MDRTBII for the different mutations. Deeplex-MycTB, GenoTypeMTBDRplusv2.0, and GenoscholarNTM+MDRTBII provide explicit information on rifampin heteroresistance for the most frequently detected mutations. Classic Xpert and Ultra report rifampin heteroresistance as rifampin resistance, while Ultra may denote rifampin heteroresistance through “mixed patterns” of wild-type and mutant melt probe, melt peak temperatures. Overall, our findings inform end users that the threshold for reporting resistance in the case of rifampin heteroresistance is the highest for Classic Xpert and Ultra to resolve phenotypic and genotypic discordant rifampin-resistant TB results.

Purpose: To investigate bacillus Calmette–Guerin (BCG) infective capability and cytotoxicity in ARPE-19 cells.Methods: BCG inoculum was dispensed at a MOI 100:1 for 3 h in 90% confluent ARPE-19 cells. Infection rates at different time points were determined by colony forming units (CFU) count and, in parallel, by the number of microscopically infected cells. WST-1 reagent was used for cytotoxicity assays.Results: A 67-year-old man previously treated with intravesical BCG for bladder carcinoma presented with chronic, refractory, bilateral uveitis with macular edema. Quiescence was achieved only after commencing antituberculous treatment. BCG infection rate by two methods peaked at 48 h (16 ± 5.7% by CFU count and 40 ± 7.7% by microscopy; p = 0.058). BCG adhesion, phagocytosis, intracellular proliferation and cytolysis was observed. Cytotoxicity was minimal and did not differ from uninfected cells.Conclusions: BCG can infect at low rates and proliferate in ARPE-19 cells without toxicity in the surro...

Repurposing of drugs to treat tuberculosis (TB) has been considered an alternative to overcome the global TB epidemic, especially to combat drug-resistant forms of the disease. Mefloquine has been reported as a potent drug to kill drug-resistant strains of Mycobacterium tuberculosis. In addition, mefloquine-derived molecules have been synthesised and their effectiveness against mycobacteria has been assessed. In this work, we demonstrate for the first time the activities of mefloquine and its oxazolidine derivative compound 1E in a murine model of TB infection following administration of both drugs by the oral route. The effects of associations between mefloquine or 1E with the clinically used antituberculosis drugs isoniazid, rifampicin, ethambutol, moxifloxacin and streptomycin were also investigated. Importantly, combination of mefloquine with isoniazid and of 1E with streptomycin showed a two-fold decrease in their minimum inhibitory concentrations (MICs). Moreover, no tested combinations demonstrated antagonist interactions. Here we describe novel evidence on the activity of mefloquine and 1E against a series of quinolone-resistant M. tuberculosis strains. These data show MICs against quinolone-resistant strains (0.5–8 µg/mL) similar to or lower than those previously reported for multidrug-resistant strains. Taking these results together, we can suggest the use of mefloquine or 1E in combination with clinically available drugs, especially in the case of resistant forms of TB.

The objective of this study was to examine the in vitro synergism of three-drug combinations against Mycobacterium tuberculosis (levofloxacin/linezolid/ethambutol, levofloxacin/amikacin/ethambutol and levofloxacin/linezolid/amikacin) using the time-kill curves method. In total, 8 multidrug-resistant and 12 drug-susceptible M. tuberculosis isolates were used. Minimum inhibitory concentrations (MICs) of the isolates for each drug were determined by the proportions method. Time-kill curves were studied for the three combinations proposed over 14 days using two different protocols. In protocol 1, 0.5× MIC for each drug was used. In protocol 2, 0.5× MIC for levofloxacin and linezolid and 0.25× MIC for amikacin and ethambutol were used. The MICs for all of the isolates studied were 0.5 mg/L for levofloxacin and linezolid and 2.5 mg/L for ethambutol and amikacin. All of the combinations displayed an additive activity compared with the most active individual drug. In conclusion, these results demonstrate that the three combinations tested were equally effective against M. tuberculosis isolates. The study of antituberculous combinations using in vitro methods is an excellent first step to predict their effect in clinical development phases as well as to test new regimens of the antituberculous drugs currently available.

Multidrug resistance has become a problem in the management of tuberculosis, with an urgent need for research into new drugs as well as the development of efficacious drug combinations and regimens. The main objective of this study was to assess and compare the efficacy of three antituberculous combinations (clofazimine/pretomanid/levofloxacin, clofazimine/pretomanid/moxifloxacin and clofazimine/pretomanid/UB-8902) against multidrug-resistant (MDR) and drug-susceptible clinical isolates of Mycobacterium tuberculosis using an in vitro adaptation of the chequerboard assay. A total of 7 MDR and 11 drug-susceptible clinical isolates were studied. The fractional inhibitory concentration index (FICI) was interpreted as synergism when the value was0.75, antagonism when it was4 and additive activity between these two values. The FICI of all of the combinations ranged from 1.2 to 2.3, showing additive activity against all of the isolates. No differences were found between MDR and drug-susceptible isolates. In conclusion, the three combinations are effective against M. tuberculosis with equal effects. Moreover, in vitro testing of drug combinations could be useful to predict their clinical use.

Purpose: To evaluate clinical and paraclinical parameters for the indirect diagnosis of tuberculosis-related uveitis (TRU). Methods: Prospective 2-year study in a tertiary referral centre. Patients with clinically suspected TRU were recruited. Demographical and clinical data were recorded. QuantiFERON®-TB Gold (QFT), tuberculosis skin test (TST) and pulmonary X-ray were performed, and other possible uveitis aetiologies were ruled out. Further investigations were also performed case by case after consultation. After final assessment, standard antituberculosis therapy was started if TRU was considered highly probable. Finally, diagnosis of TRU was established according to current criteria and set as gold standard. Strength of association for TRU was determined by odds ratio and compared by appropriate tests. Concordance and binary classification tests were also assessed. Results: The study included 103 patients, 54 men and 49 women. Mean age 45.6 years. Sixty-eight patients were Spanish-born and 35 were foreign-born. Final diagnosis included 33 (32%) cases of TRU and 70 (67%) cases with other diagnoses. Asian origin (OR = 3.50, p = 0.046), previous tuberculosis (TB) contact (OR = 2.61, p = 0.026), TB in the past (OR = 6.18, p = 0.004) and associated retinal vasculitis (OR = 7.85, p

The aim of this study was to evaluate the reliability of the VersaTREK system for Mycobacterium tuberculosis drug susceptibility testing compared with results obtained with the Bactec MGIT 960 system. A total of 67 strains were evaluated. Overall agreement was at 98.5%. Kappa indexes were 1.0 for isoniazid, rifampin, and ethambutol, 0.937 for pyrazinamide, and 0.907 for streptomycin. The VersaTREK system is validated for M. tuberculosis drug susceptibility testing.

A diagnostic protocol was started to study the etiology of pulmonary infiltrates in immunosuppressed patients. The diagnostic yields of the different techniques were analyzed, with special emphasis on the importance of the sample quality and the role of rapid techniques in the diagnostic strategy. In total, 241 patients with newly developed pulmonary infiltrates within a period of 19 months were included. Noninvasive or invasive evaluation was performed according to the characteristics of the infiltrates. Diagnosis was achieved in 202 patients (84%); 173 patients (72%) had pneumonia, and specific etiologic agents were found in 114 (66%). Bronchoaspirate and bronchoalveolar lavage showed the highest yields, either on global analysis (23 of 35 specimens [66%] and 70 of 134 specimens [52%], respectively) or on analysis of each type of pneumonia. A tendency toward better results with optimal-quality samples was observed, and a statistically significant difference was found in sputum bacterial culture. Rapid diagnostic tests yielded results in 71 of 114 (62.2%) diagnoses of etiological pneumonia.

Purpose To report the anatomical pattern and etiological spectrum of uveitis in an urban multi-ethnic population from Barcelona, Spain. General and specific epidemiological data for the most prevalent aetiologies are also calculated. Methods A cross-sectional study of consecutive uveitis cases was performed between 1 January 2009 and 31 December 2012. Exogenous endophthalmitis, surgery-related, post-traumatic and toxic uveitis along with masquerade syndromes were excluded. Anatomical (Standard Uveitis Nomenclature criteria) and aetiological patterns (by tailored tests), age, sex, geographical origin and laterality were analysed. Mean incidence and prevalence were calculated for a mid-period reference population. Results From 1022 patients included, 52% were anterior uveitis (AU), 23% posterior, 15% panuveitis and 9% intermediate uveitis. Aetiologically, 26% were unclassifiable, 29% infectious, 25% associated with systemic immune diseases, and 20% corresponded to ocular-specific syndromes. Among classified causes, herpesvirus (12%), toxoplasma (7%), Behcet's disease (BD) (5%), HLA-B27-isolated AU (5%), ankylosing spondylitis (5%), tuberculosis-related uveitis (TRU) (5%), birdshot chorioretinopathy (3%) and sarcoidosis (3%) were the most frequent. Non-Spanish origin was recorded in 22%, with 47% of Vogt-Koyanagi-Harada and 36% of toxoplasma cases coming from South America, 10% of BD and 11% of TRU from Africa and 24% of TRU cases from Asia. A mean annual incidence of 51.91 cases/100 000 inhabitants was found for the referral population. Conclusion In our referral area, 74% of the uveitis cases can be correctly classified. A large myriad of uveitis aetiologies with a strong geographical origin burden are found in Western urban multi-ethnic populations.

Multidrug resistance is a problem in the management of tuberculosis, creating an urgent need for new regimens including currently available drugs. Macrophage models allow an evaluation of the effect of drugs against intracellular bacilli. The effect of the following different drug combinations against six multidrug-resistant and six drug-susceptible clinical isolates of Mycobacterium tuberculosis multiplying inside the human macrophage THP-1 cell line was studied: levofloxacin/linezolid/ethambutol; levofloxacin/amikacin/ethambutol; and levofloxacin/linezolid/amikacin. Macrophages were lysed, seeded onto Middlebrook 7H11 plates and CFU were counted after 21 days of incubation. The interaction of the drugs in combination was interpreted by the effect of the combination compared with the most active single drug alone. The antimicrobial activity of the drugs was evaluated comparing the log(10)CFU/well of the isolate with and without the drug. Drug concentrations within infected macrophages and in extracellular medium were simultaneously determined by chromatography. The levofloxacin/linezolid/amikacin and levofloxacin/linezolid/ethambutol combinations showed antagonism against most of the isolates (91.7%) after a 4-day protocol, whereas levofloxacin/amikacin/ethambutol displayed indifference. Levofloxacin alone and levofloxacin/amikacin/ethambutol were the most potent antimicrobials, presenting reductions up to 5.49 log(10) and 5.86 log(10), respectively. The drug penetration percentages ranged from 5.46% to 11.10%. Intracellular concentrations for the drug alone compared with those for the drugs in combination were not significantly different. All of the combinations tested against M. tuberculosis-infected macrophages showed antimicrobial activity, with combinations including linezolid and levofloxacin showing an antagonistic effect that may be explained by efflux transporters or changes in the macrophage environment.

Objective To evaluate the resistance and mortality rates of multidrug-resistant tuberculosis (MDR-TB) in human immunodeficiency virus type 1 (HIV-1)-infected patients in a 1000-bed teaching hospital in inner-city Barcelona. Methods A total of 311 patients, recruited between July 1991 and July 1993, had either tuberculosis (TB) with HIV (group A), TB without HIV (group B), or TB without HIV and previous exposure to antituberculosis drugs (group C). Mycobacterium tuberculosis was isolated radiometrically or using Lowenstein-Jensen media with p-nitro-a-acetylamino-B-hidroxypropiphenone (NAP) testing. Antibiograms to isoniazid, rifampicin, pyrazinamide, ethambutol and streptomycin were performed by the critical proportions method with only one strain per patient. Results Of 197 strains of M. tuberculosis isolated in group A, six (3%) were resistant to one agent and seven (3.5%) were multiresistant vs five (5.7%) and three (3.4%) of 88 strains, respectively, in group B, and three (11.5%) and seven (26.9%) of 26 strains, respectively, in group C. Conclusions The primary resistance rate of M. tuberculosis in Spain remains relatively low in both HIV-infected and non-HIV-infected patients in contrast to a nearly 30% or higher resistance rate among patients previously exposed to antituberculosis agents.

Summary Fluoroquinolones such as ofloxacin are promising drugs to treat drug-resistant tuberculosis (TB) and have been proposed for shortening the treatment of TB. The objectives were to study the synergistic effect of the combinations of three drugs and to evaluate the in vitro interactions of the following combinations against Mycobacterium tuberculosis : A) isoniazid, rifampicin, and ethambutol and B) ofloxacin, rifampicin, and ethambutol using an adaptation of the two-dimensional chequerboard assay. A total of 12 isolates resistant to isoniazid or to isoniazid-streptomycin and 11 drug-susceptible isolates were tested. The fractional inhibitory concentration (FICI) was calculated as follows: FICI = FIC A + FICB + FIC C = A/MIC A + B/MIC B + C/MIC C where A, B and C were the MICs of each antibiotic in combination and MIC A , MIC B and MIC C were the individual MICs. The FICI was interpreted as synergism when the value was ≤0.75. In combination A, 11 drug-susceptible isolates decreased the individual MIC one to three dilutions, showing indifferent activity in 81.8% (FICI = 0.88–1.6) and synergistic activity in 18.1% (FICI = 0.6). In combination B, 21 out of the 23 isolates studied (91.3%) showed synergism (FICI = 0.31–0.62). In conclusion, adaptation of the two-dimensional chequerboard assay is a reliable method to study in vitro three-drug combinations. Both three-drug combinations tested may be useful against drug-resistant isolates, although the combination including ofloxacin showed better efficacy, being of potential use in drug-susceptible and isoniazid-resistant isolates.

During a 2-year period (2003–2004), tuberculosis (TB) transmission in Barcelona and the factors related to transmission among the Spanish- and foreign-born populations were studied by molecular epidemiology. Data were obtained from TB cases and Conventional Contact Tracing registries and genotyping was performed using restriction fragment length polymorphism (RFLP)-IS6110 and MIRU12 as a secondary typing method. Of the 892 TB cases reported, 583 (65.3%) corresponded to Spanish-born and 309 (34.6%) to foreign-born. Six hundred and eighty-seven cases (77%) were confirmed by culture. RFLP typing of 463/687 (67.4%) isolates was performed, revealing 280 (60.5%) unique and 183 (39.5%) shared patterns, which were grouped into 65 clusters. Spanish-born individuals were significantly more clustered than foreign-born individuals (44.6% vs. 28.8%; p 0.016). Clustering in foreign-born individuals was associated with HIV (p 0.051, odds ratio = 3.1, 95% confidence interval 1–10.9) and alcohol abuse (p 0.022), whereas, in the Spanish-born individuals, clustering was associated with age in the range 21–50 years, (p 0.024). Of the total clusters, 36/65 (55.3%) included only Spanish-born patients, whereas 22/65 (33.8%) included individuals from both populations. In mixed clusters, the index case was Spanish-born in 53% and foreign-born in 47%. Among the foreign-born, 2.8% were ill on arrival, 30% developed TB within the first year and 50.3% developed TB within the first 2 years; 58.3% were from South America. In conclusion, half of the foreign-born TB patients developed the disease during the first 2 years after arrival, which, in most cases, was the result of endogenous reactivation. Recent TB transmission among Spanish-born and foreign-born populations, as well as bidirectional transmission between communities, contributed significantly to the burden of TB in Barcelona, suggesting the need to improve Public Health interventions in both populations.

In this multicenter study, the reliability of two nonradiometric, fully automated systems, the MB/BacT and BACTEC MGIT 960 systems, for testing the susceptibilities of 82 Mycobacterium tuberculosis strains to isoniazid, rifampin, ethambutol, and streptomycin was evaluated in comparison with the radiometric BACTEC 460TB system. The arbitration of discrepant results was done by the reanalysis of the strain, the determination of the MIC, and the molecular characterization of some resistance determinants. The overall level of agreement with BACTEC 460TB results was 96% with the MB/BacT test and 97.2% with the BACTEC MGIT 960 system. With both methods, the level of agreement with BACTEC 460TB results was 96.3% for isoniazid, 98.8% for rifampin, and 98.8% for ethambutol. The level of agreement for streptomycin was 90.2% with MB/BacT and 97.5% with BACTEC MGIT 960. Overall, there were 11 very major errors and 2 major errors with the MB/BacT method and 5 very major errors and 2 major errors with the BACTEC MGIT 960 system. In general, the MB/BacT and BACTEC MGIT 960 systems showed good performance for susceptibility testing with first-line antituberculosis drugs.