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Background: Negative (vacuum) pressure therapy promotes wound healing. However, commercially available devices are unaffordable to most potential users in low- and middle-income countries (LMICs), limiting access to many patients who could benefit from this treatment. This study aimed to design and test a cheap and easy-to-build negative pressure device and provide its detailed open-source description, thereby enabling free replication. Methods: the negative pressure device was built using off-the-shelf materials available via e-commerce and was based on a small pump, a pressure transducer, and the simplest Arduino controller with a digital display (total retail cost ≤ 75 US$). The device allows the user to set any therapeutic range of intermittent negative pressure and has two independent safety mechanisms. The performance of the low-cost device was carefully tested on the bench using a phantom wound, producing a realistic exudate flow rate. Results: the device generates the pressure patterns set by the user (25–175 mmHg of vacuum pressure, 0–60 min periods) and can drain exudate flows within the clinical range (up to 1 L/h). Conclusions: a novel, low-cost, easy-to-build negative pressure device for wound healing displays excellent technical performance. The open-source hardware description provided here, which allows for free replication and use in LMICs, will facilitate the application and wider utilization of this therapy to patients.

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 ...

The 2-(quinolin-4-yloxy)acetamides (QOAs) have been reported to be promising molecules for tuberculosis treatment. Recent studies demonstrated their potent antimycobacterial activity, biological stability and synergism with rifampicin. The identification of the molecular target is an essential step towards the development of a novel drug candidate. Here, we report the target identification of the QOAs. We found that these compounds are active against Mycobacterium tuberculosis clinical isolates resistant to isoniazid, rifampicin, ethambutol, streptomycin and ethionamide. The initial evidence that DNA gyrase might be the target of QOAs, based on high minimum inhibitory concentration (MIC) values against ofloxacin-resistant clinical isolates and structural similarities with fluoroquinolones, was discarded by experiments performed with M. tuberculosis GyrA point mutant, DNA gyrase supercoiling inhibition assay and overexpression of DNA gyrase. We selected spontaneous mutants for our lead compound 21 and observed that these strains were also resistant to all QOA derivatives. The genomes of the spontaneous mutants were sequenced, and the results revealed a single mutation in qcrB gene (T313A), which indicates that the QOAs target the cytochrome bc1 complex. The protein-compound interaction was further investigated by molecular docking. These findings reinforce the relevance of these compounds as promising candidates for the treatment of multidrug-resistant tuberculosis.

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.

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.

The aim of this study was to analyze the factors associated with conventional contact tracing (CCT) and molecular epidemiology (ME) methods in assessing tuberculosis (TB) transmission, comparing the populations studied and the epidemiological links established by both methods. Data were obtained from TB case and CCT registries, and ME was performed using IS 6110 -based restriction fragment length polymorphism (RFLP) analysis and mycobacterial interspersed repetitive unit 12 (MIRU12) typing as a secondary typing method. During two years (2003 and 2004), 892 cases of TB were reported, of which 687 (77%) were confirmed by culture. RFLP analysis was performed with 463 (67.4%) of the 687 isolated strains, and MIRU12 types in 75 strains were evaluated; 280 strains (60.5%) had a unique RFLP pattern, and 183 (39.5%) shared patterns, grouping into 65 clusters. CCT of 613 (68.7%) of 892 cases detected 44 clusters involving 101 patients. The results of both CCT and ME methods yielded 96 clusters involving 255 patients. The household link was the one most frequently identified by CCT (corresponding to 80.7% of the cases clustered by this method), whereas nonhousehold and unknown links were associated with 94.1% of the strains clustered by ME. When both methods were used in 351 cases (39.3%), they showed the same results in 214 cases (61%). Of the remainder, 106 (30.2%) were clustered only by ME, 19 (5.5%) were clustered only by CCT, and 12 (3.4%) were clustered by both methods but into different clusters. Patients with factors potentially associated with social problems were less frequently studied by CCT ( P = 0.002), whereas patients of P = 0.005). Significant differences in the populations studied by ME versus CCT were observed, possibly explaining the scarce correlation found between the results of these methods. Moreover, ME allowed the detection of nonhousehold contact relationships, whereas CCT was more useful for tracing transmission chains involving patients of

A total of 243 clinical isolates of the Mycobacterium genus were studied, 143 and 100 using two protocols (Protocol v2 and Protocol v3, respectively) provided by the manufacturer. The overall correlation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with the standard identification methods was 63.8 %. The rate of misidentification was 3.2 %, mainly affecting very close species. In Protocol v2, the correlation was 57.3 %, being greater in solid than in liquid media (71.7 % vs. 44.7 %, p

Early diagnosis of tuberculosis and screening of other mycobacteria is required for the appropriate management of patients. We have therefore developed a 5′-exonuclease fluorogenic PCR assay in a single-tube balanced heminested format that simultaneously detects Mycobacterium tuberculosis complex (MTC) and members of the Mycobacterium genus (MYC) using the 16S ribosomal DNA target directly on clinical samples. One hundred twenty-seven clinical samples (65 smear negative and 62 smear positive) with a positive culture result from 127 patients were tested, including 40 negative control specimens. The finding of both a positive MTC and probe value and a positive MYC probe value confirmed the presence of MTC or mycobacteria with a 100% positive predictive value. However, a negative value for MTC or MYC did not discount the presence of mycobacteria in the specimen. Interestingly, the addition of the MYC probe allowed the diagnosis of an additional 7% of patients with tuberculosis and rapid screening of nontuberculous mycobacteria (NTM). Thus, over 75% of the patients were diagnosed with mycobacterial disease by PCR. The sensitivity was much higher on smear-positive samples (90.3%) than smear-negative samples (49.2%) and was slightly higher for MTC than NTM samples. With regard to the origin of the sample, MTC pulmonary samples gave better results than others. In conclusion, we believe this test may be useful for the rapid detection of mycobacteria in clinical samples and may be a valuable tool when used together with conventional methods and the clinical data available.

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.

Ethambutol resistance has mostly been related to mutations in the embB gene. The objective of the present study was to characterize the embB gene in a collection of ethambutol-resistant and ethambutol-susceptible isolates of Mycobacterium tuberculosis complex (MTBC) from Barcelona, and to develop a DNA microarray for the rapid detection of embB mutations in our area. Fifty-three ethambutol-resistant and 702 ethambutol-susceptible isolates of MTBC were sequenced in internal 9821495 bp fragments of the embB gene. In addition, a low-cost, low-density array was designed to include the embB codons identified as being most frequently mutated in our area (LD-EMB array). The global prevalence of embB mutations found among the ethambutol-resistant isolates was 77.4 (41/53). Substitutions in embB306 were the most common [53.7 (22/41)], followed by substitutions in embB406 [26.8 (11/41)]. The presence of mutations in embB406 was related to higher levels of ethambutol resistance and to multidrug resistance. Among unrelated isolates (from 24-locus MIRU-VNTR genotyping), the percentage of embB-mutated isolates was 72.9 (27/37)u59.3 (16/27) in embB306 and 25.9 (7/27) in embB406. None of the ethambutol-susceptible isolates studied showed a mutation in codon 306 or 406. The LD-EMB array showed 100 sensitivity and specificity in identifying the main embB substitutions in our area. Mutations at codons 306 and 406 of embB have a relevant role in resistance to ethambutol in our area. The LD-EMB array developed in this study would appear to be a good molecular test for rapid detection of ethambutol resistance.

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.

Mycobacterium sherrisii is a new species of opportunistic, slow-growing, non-tuberculous Mycobacterium closely related to Mycobacterium simiae that can currently be identified with the sequence of 16S rARN gene and the heat-shock protein 65. Few cases of patients infected by this Mycobacterium have been reported and all of them were associated with human immunodeficiency virus or other immunosuppressive conditions. Clinical management is complex, because there is not a clear correlation between the in vitro antibiotic susceptibility testing and the patient's clinical outcome.

In cases of multidrug-resistant tuberculosis, it is crucial to rule out resistance to second-line antituberculous (anti-TB) agents. In the present study, a low-cost low-density DNA array including four genetic regions (rrs 530 loop, rrs 1400, rpsL and gyrA) was designed for the rapid detection of the most important mutations related to anti-TB injectable drugs (mainly streptomycin) and fluoroquinolone resistance (LD-SQ array). A total of 108 streptomycin- and/or ofloxacin-resistant and 20 streptomycin- and ofloxacin-susceptible Mycobacterium tuberculosis clinical isolates were analysed with the array. The results obtained were compared with sequencing data and phenotypic susceptibility pattern. The LD-SQ array offered a good sensitivity compared to sequencing, especially among resistant strains: 92.5% (37/40) for streptomycin and 87.5% (7/8) for fluoroquinolones. Therefore, this array could be considered a good approach for the rapid detection of mutations related to streptomycin and fluoroquinolone resistance. On the other hand, there were discordant results in 16 resistant strains and six susceptible isolates, mostly concerning the gyrA region, in which the existence of polymorphisms next to informative positions might cause cross-hybridization. These discrepancies were caused by some technical limitations; consequently, the present array should be considered as a first-step prior to a forthcoming optimized version of the array. (c) 2013 Elsevier Ltd. All rights reserved.

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.

Multidrug resistance has become a problem in the management of tuberculosis, leading to an urgent need for research related to new regimens including the currently available drugs. The objectives of this study were: (i) to study the effect of the following second-choice three-drug combinations against multidrug-resistant (MDR) and drug-susceptible clinical isolates (levofloxacin, linezolid and ethambutol; levofloxacin, amikacin and ethambutol; and levofloxacin, linezolid and amikacin); and (ii) to compare the effect of these combinations with an isoniazid, rifampicin and ethambutol combination against drug-susceptible clinical isolates. A total of 9 MDR clinical and 12 drug-susceptible isolates (11 clinical isolates and the H37Rv reference strain) were studied using an adaptation of the chequerboard assay. The fractional inhibitory concentration index (FICI) was calculated as follows: FICI=FIC(A)+FIC(B)+FIC(C)=A/MIC(A)+B/MIC(B)+C/MIC(C), where A, B and C are the minimum inhibitory concentrations (MICs) of each antibiotic in combination and MIC(A), MIC(B) and MIC(C) are the individual MICs. The FICI was interpreted as synergism when the value was

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.

The purpose of the study was to compare the IS6110-RFLP (RFLP) results obtained in a previous epidemiological study in the city of Barcelona, Spain [Borrell S, Espanol M, Orcau A, Tudo G, March F, Cayla JA, et al. Factors associated with differences between conventional contact tracing and molecular epidemiology in study of tuberculosis transmission and analysis in the city of Barcelona, Spain. J Clin Microbial 2009 Jan;47(1):198-204.] with the results obtained with IS6110-fAFLP, [Thorne N, Evans JT, Smith EG, Hawkey PM, Charbia S, Arnold C. An IS6110-targeting fluorescent amplified fragment length polymorphism alternative to IS6110-restriction fragment length polymorphism analysis for Mycobacterium tuberculosis DNA fingerprinting. Clin Microbiol Infect 2007 Oct;13(10):964-70.] on the ability to detect recent transmission. fAFLP was applied to DNA samples of RFLP clustered strains, with and without known epidemiological links, with the additional inclusion of four nucleotide-specific fluorophores to further increase the discrimination of the fragments obtained. Four-colour fAFLP was performed on 123 RFLP clustered strains with no epidemiological link (NELC) and on 28 epidemiologically linked RFLP clustered (ELC) strains grouped into 48 and 13 clusters respectively. Clustering results obtained by the two methods were highly congruent in ELC strains with fAFLP allocating 92.3% of the ELCs. For the NELCs, RFLP results were confirmed in 39/48 (81.2%) of fAFLP-clusters with 0-1 different fragments and 9/48 (18.8%) differed in 2-4 fragments, which are considered genetically related but not recently transmitted. In conclusion, overestimation of recent tuberculosis transmission can occur because of the inaccurate analysis of RFLP results. Four-colour fAFLP allows us to differentiate between recent transmission strains and epidemiologically unrelated but genetically related strains. (C) 2009 Elsevier Ltd. All rights reserved.

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.

Background: This study evaluates a method based on real-time PCR for direct detection in clinical samples of the common mutations responsible for isoniazid and rifampicin resistance of Mycobacterium tuberculosis. Methods: Six pairs of fluorogenic 5 0 exonuclease probes (Taqmanw), mutated and wild-type, were designed for six targets: codon 315 of katG, substitution C209T in the regulatory region of inhA, and codons 513, 516, 526 and 531 of rpoB. Results: A total of 98 clinical samples harbouring resistant bacilli from 55 patients and 126 samples harbouring susceptible bacilli from 126 patients were processed. The isolates from samples were tested for drug susceptibility with the radiometric method and sequenced for the same genetic targets. Among the samples, 93 harboured isoniazid-resistant bacilli. According to the sequencing results, 30 had mutations in katG, 30 in inhA and 33 (35.4%) had no mutations in these targets. All 27 clinical specimens harbouring rifampicin-resistant bacilli showed mutations in rpoB. The detection threshold of this method in detecting target genes in serial dilutions of artificial samples was 1.5 3 10 3 cfu/mL. In clinical samples, the sensitivity ranged from 30.4 to 35.3% for smear-negative samples and from 95.1 to 99.2% for smear-positive samples, with a specificity of 100%. In this study, the overall sensitivity in detecting patients having the target mutations was 74.3%. Conclusions: The main advantage of the described method is the possibility of detecting rifampicin and isoniazid resistance within 48‐72h after sample collection, with a sensitivity of nearly 100% in smear-positive samples if the chosen target is responsible for the resistance.