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Whole genome sequencing (WGS) is revolutionary for diagnostics of TB and its mutations associated with drug-resistances, but its uptake in low- and middle-income countries is hindered by concerns of implementation feasibility. Here, we provide a proof of concept for its successful implementation in such a setting. WGS was implemented in the Kyrgyz Republic. We estimated needs of up to 55 TB-WGS per week and chose the MiSeq platform (Illumina, USA) because of its capacity of up to 60 TB-WGS per week. The project’s timeline was completed in 93-weeks. Costs of large equipment and accompanying costs were 222,065 USD and 8462 USD, respectively. The first 174 WGS costed 277 USD per sequence, but this was skewed by training inefficiencies. Based on real prices and presuming optimal utilization of WGS capacities, WGS costs could drop to 167 and 141 USD per WGS using MiSeq Reagent Kits v2 (500-cycles) and v3 (600-cycles), respectively. Five trainings were required to prepare the staff for autonomous WGS which cost 48,250 USD. External assessment confirmed excellent performance of WGS by the Kyrgyz laboratory in an interlaboratory comparison of 30 M. tuberculosis genomes showing complete agreeance of results.

Xpert MTB/RIF testing has improved tuberculosis (TB) diagnostics and rifampicin (Rif) resistance testing worldwide. However, it has weaknesses, such as its restriction to Rif resistance testing and the inability to use extracted DNA for further testing. Herein, a holistic diagnostic workflow, including TB detection and resistance testing toward Rif, isoniazid, and important second-line drugs (SLDs), based on a novel microfluidic DNA extraction cartridge (TB-Disk), is presented. DNA from 73 precharacterized sputum samples was extracted with TB-Disk, including 45 clinical and bacteriologically confirmed TB samples, nine TB-negative samples, and 19 sputum samples spiked with twofold dilutions of TB bacteria. The extracted DNA was subjected to further testing with FluoroType MTB (FT-MTB), GenoType MTBDRplus (GT-plus), and GenoType MTBDRsl. A total of 100% (20/20) and 72% (18/25) of smear-positive and smear-negative TB samples were identified as Mycobacterium tuberculosis complex positive. A total of 79% (33/42) of subsequently GT-plus tested samples yielded a valid result. Eight samples were identified as multidrug-resistant TB by GT-plus and further tested for resistance toward SLDs using GenoType MTBDRsl, yielding 75% (6/8) valid results. FT-MTB with cartridge-based DNA extraction (Disk-DNA) and DNA extracted with FluoroLyse yielded similar analytical sensitivities. FT-MTB with Disk-DNA was 100% specific. TB-Disk in combination with FT-MTB enables sensitive TB detection. The Disk-DNA can be further used for screening resistance toward first-line drugs and SLDs.

We present a novel centrifugal microfluidic approach for fast and accurate tuberculosis (TB) diagnosis based on the use of standard laboratory equipment. The herein presented workflow can directly be integrated into laboratories with standard equipment and automates complex sample preparation. The system consists of a microfluidic cartridge, a laboratory centrifuge and a standard PCR cycler. The cartridge includes all required reagents and automates collection of bacteria on filter membranes, bacterial lysis, nucleic acid extraction and aliquoting of the DNA extract for PCR analysis. We show that storage of the reagents in aluminium-coated pouches is stable during accelerated storage and transport tests. When the limit of detection was assessed, we found that the cartridge-automated workflow consistently detected 10 CFU ml-1 of mycobacteria in spiked sputum samples. First tests with clinical samples showed a 100% specificity for non-TB specimens. In addition, Mycobacterium tuberculosis (MTB) was re-found in pre-characterized smear microscopy and culture positive sputum samples suggesting a high diagnostic sensitvity. In summary, the novel cartridge-automated workflow enables a flexible and sensitive TB diagnosis without the need to invest in specialized instrumentation.

Background Effective active case finding (ACF) activities are essential for early identification of new cases of active tuberculosis (TB) and latent TB infection (LTBI). Accurate diagnostics as well as the ability to identify contacts at high risk of infection are essential for ACF, and have not been systematically reported from Central Asia. The objective was to implement a pilot ACF program to determine the prevalence and risk factors for LTBI and active TB among contacts of individuals with TB in Kyrgyz Republic using Quantiferon-TB Gold plus (QuantiFERON). Methods An enhanced ACF project in the Kyrgyz Republic was implemented in which close and household (home) contacts of TB patients from the Issyk-Kul Oblast TB Center were visited at home. QuantiFERON and the tuberculin skin test (TST) alongside clinical and bacteriological examination were used to identify LTBI and active TB cases among contacts. The association for QuantiFERON positivity and risk factors were analysed and compared to TST results. Results Implementation of ACF with QuantiFERON involved close collaboration with the national sanitary and epidemiological services (SES) and laboratories in the Kyrgyz Republic. From 67 index cases, 296 contacts were enrolled of whom 253 had QuantiFERON or TST results; of those 103 contacts had LTBI (positive TST or IGRA), and four (1.4%) active TB cases were detected. Index case smear microscopy (OR 1.76) and high household density (OR 1.97) were significant risk factors for QuantiFERON positivity for all contacts. When stratified by age, association with smear positivity disappeared for children below 15 years. TST was not associated with any risk factor. Conclusions This is the first time that ACF activities have been reported for Central Asia, and provide insight for implementation of effective ACF in the region. These ACF activities using QuantiFERON led to increase in the detection of LTBI and active cases, prior to patients seeking treatment. Household density should be taken into consideration as an important risk factor for the stratification of future ACF activities.

BD MAX MDR-TB assay is a new molecular platform for the detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens and simultaneous detection of resistance towards isoniazid and rifampicin. In this study, we assessed its diagnostic accuracy using pre-characterized MTBC-culture negative (n=257), smear-negative / MTBC-culture positive (n=93), and smear-positive / MTBC-culture positive (n=153) respiratory specimens. Compared to culture, overall sensitivity and specificity of BD MAX MDR-TB were 86.6% and 100%, respectively; sensitivities for smear-positive and smear-negative samples were 100% and 64.5%, respectively. Sensitivity and specificity for isoniazid and rifampicin resistance were 58.3% (biased low due to sample collection strategy in low prevalence setting), 99.3%, 100%, and 98.2%, respectively, compared to phenotypic drug resistance testing and 100%, 99.4%, 100% and 99.4%, respectively, compared to Genotype MTBDRplus. In conclusion, BD MAX MDR-TB is an accurate assay for the diagnostic detection of MTBC in respiratory samples and its resistance toward the most important anti-TB drugs isoniazid and rifampicin. Due to its medium to high throughput, good validity and ease of use, the assay will be of great benefit for medium-size to large TB diagnostic centers.

Whole-genome sequencing (WGS) of Mycobacterium tuberculosis complex has become an important tool in diagnosis and management of drug-resistant tuberculosis. However, data correlating resistance genotype with quantitative phenotypic antimicrobial susceptibility testing (AST) are scarce.In a prospective multicentre observational study, 900 clinical M tuberculosis complex isolates were collected from adults with drug-resistant tuberculosis in five high-endemic tuberculosis settings around the world (Georgia, Moldova, Peru, South Africa, and Viet Nam) between Dec 5, 2014, and Dec 12, 2017. Minimum inhibitory concentrations (MICs) and resulting binary phenotypic AST results for up to nine antituberculosis drugs were determined and correlated with resistance-conferring mutations identified by WGS.Considering WHO-endorsed critical concentrations as reference, WGS had high accuracy for prediction of resistance to isoniazid (sensitivity 98·8% [95% CI 98·5-99·0]; specificity 96·6% [95% CI 95·2-97·9]), levofloxacin (sensitivity 94·8% [93·3-97·6]; specificity 97·1% [96·7-97·6]), kanamycin (sensitivity 96·1% [95·4-96·8]; specificity 95·0% [94·4-95·7]), amikacin (sensitivity 97·2% [96·4-98·1]; specificity 98·6% [98·3-98·9]), and capreomycin (sensitivity 93·1% [90·0-96·3]; specificity 98·3% [98·0-98·7]). For rifampicin, pyrazinamide, and ethambutol, the specificity of resistance prediction was suboptimal (64·0% [61·0-67·1], 83·8% [81·0-86·5], and 40·1% [37·4-42·9], respectively). Specificity for rifampicin increased to 83·9% when borderline mutations with MICs overlapping with the critical concentration were excluded. Consequently, we highlighted mutations in M tuberculosis complex isolates that are often falsely identified as susceptible by phenotypic AST, and we identified potential novel resistance-conferring mutations.The combined analysis of mutations and quantitative phenotypes shows the potential of WGS to produce a refined interpretation of resistance, which is needed for individualised therapy, and eventually could allow differential drug dosing. However, variability of MIC data for some M tuberculosis complex isolates carrying identical mutations also reveals limitations of our understanding of the genotype and phenotype relationships (eg, including epistasis and strain genetic background).BillMelinda Gates Foundation, German Centre for Infection Research, German Research Foundation, Excellence Cluster Precision Medicine of Inflammation (EXC 2167), and Leibniz ScienceCampus EvoLUNG.

BACKGROUND Abbott RealTime MTB RIF/INH Resistance (RT RIF/INH) is a new assay for the detection of resistance to rifampicin (RIF) and isoniazid (INH) in tuberculosis (TB) patients. OBJECTIVE To evaluate the capacity of RT RIF/INH to detect resistance-associated mutations in target genes. METHODS A total of 311 Mycobacterium tuberculosis strains that had been pre-characterised using genotypic methods (GenoType® MTBDRplus, Sanger sequencing) and phenotypic drug susceptibility testing were subjected to DNA extraction on Abbott m2000sp and analysed using RT RIF/INH. Detection of heteroresistant mutations was studied with artificial mixtures of wild-type and mutant DNA. RESULTS Overall sensitivity and specificity values of RT RIF/INH to detect resistance were respectively 87.2% and 98.4% for RIF and respectively 90.1% and 99.2% for INH. The capacity of RT RIF/INH to detect specific mutations was 100% for katG, inhA and frequent rpoB mutations, and 76% for rare rpoB mutations. Among the latter, two rare mutations were not consistently detected. With heteroresistant samples, RT RIF/INH reported resistance if samples contained at least 75-90% of mutant DNA. CONCLUSION RT RIF/INH is a reliable high-throughput assay for the detection of RIF and INH resistance markers. The ability to detect INH resistance also may be of benefit in areas with high rates of INH-resistant, non-multidrug-resistant TB. .

M. tuberculosis grows slowly and is challenging to work with experimentally compared with many other bacteria. Although microtitre plates have the potential to enable high-throughput phenotypic testing of M. tuberculosis, they can be difficult to read and interpret. Here we present a software package, the Automated Mycobacterial Growth Detection Algorithm (AMyGDA), that measures how much M. tuberculosis is growing in each well of a 96-well microtitre plate. The plate used here has serial dilutions of 14 anti-tuberculosis drugs, thereby permitting the MICs to be elucidated. The three participating laboratories each inoculated 38 96-well plates with 15 known M. tuberculosis strains (including the standard H37Rv reference strain) and, after 2 weeks' incubation, measured the MICs for all 14 drugs on each plate and took a photograph. By analysing the images, we demonstrate that AMyGDA is reproducible, and that the MICs measured are comparable to those measured by a laboratory scientist. The AMyGDA software will be used by the Comprehensive Resistance Prediction for Tuberculosis: an International Consortium (CRyPTIC) to measure the drug susceptibility profile of a large number (>30000) of samples of M. tuberculosis from patients over the next few years.

Background: Health care workers (HCW) are at increased risk of TB infection due to their close contact with infected patients with active TB. The objectives of the study were (1) to assess the prevalence of LTBI among HCW in major TB hospitals of the Northern Kyrgyz Republic, and (2) to determine the association of LTBI with certain job positions, hospital units, or hospitals. Methods: HCWs from 4 TB hospitals in the Northern Kyrgyz Republic were tested with the interferon-gamma release assay (IGRA) Quantiferon-TB Gold plus (QFT) for the detection of an immune response to TB as marker of TB infection. Age was controlled for as a confounder. Univariate and multivariable analysis were performed using logistic regression to assess the association of the risk factors (age, job position, and hospital unit) with having a QTF positive result. Firth’s penalized-likelihood estimates were used for determining the odd’s of QFT positivity to account for the small-sample size. Pairwise comparisons using the Bonferroni correction (conservative) and comparisons without adjusting for multiple comparisons (unadjusted) were used to identify the categories where differences occurred.Results: QFT yielded valid results for 404 HCW, with 189 (47%) that had a positive test. In the National Tuberculosis Center, there was an increased odds to have a positive QFT test for the position of physician (OR=8.7, 95%, CI=1.2-60.5, p=0.03) and laboratory staff (OR=19.8, 95% CI=2.9-135.4, pConclusions: This is the first report on prevalence and risk factors of LTBI for HCW in the Kyrgyz republic, and results indicate an increased risk for LTBI among physicians and laboratory personnel.

Background Health care workers (HCW) are at increased risk of TB infection due to their close contact with infected patients with active TB. The objectives of the study were (1) to assess the prevalence of LTBI among HCW in the Northern Kyrgyz Republic, and (2) to determine the association of LTBI with job positions or departments. Methods HCWs from four TB hospitals in the Northern Kyrgyz Republic were tested with the interferon-gamma release assay (IGRA) Quantiferon-TB Gold plus (QFT) for the detection of an immune response to TB as marker of TB infection. Age was controlled for as a confounder. Univariate and multivariable analysis were performed using logistic regression to assess the association of the risk factors (job position, and department) with having a QTF positive result. Firth’s penalized-likelihood estimates were used to account for the small-sample size. Pairwise comparisons using the Bonferroni correction (conservative) and comparisons without adjusting for multiple comparisons (unadjusted) were used to identify the categories where differences occurred. Results QFT yielded valid results for 404 HCW, with 189 (46.7%) having a positive test. In the National Tuberculosis Center there was an increased odds to have a positive QFT test for the position of physician (OR = 8.7, 95%, CI = 1.2–60.5, p = 0.03) and laboratory staff (OR = 19.8, 95% CI = 2.9–135.4, p Conclusions This is the first report on prevalence and risk factors of LTBI for HCW in the Kyrgyz republic, and results indicate there may be an increased risk for LTBI among physicians and laboratory personnel. Further research should investigate gaps of infection control measures particularly for physicians and laboratory staff and lead to further improvement of policies.

We present a novel centrifugal microfluidic approach for fast and accurate tuberculosis (TB) diagnosis based on the use of standard laboratory equipment. The herein presented workflow can directly be integrated into laboratories with standard equipment and automates complex sample preparation. The system consists of a microfluidic cartridge, a laboratory centrifuge and a standard PCR cycler. The cartridge includes all required reagents and automates collection of bacteria on filter membranes, bacterial lysis, nucleic acid extraction and aliquoting of the DNA extract for PCR analysis. We show that storage of the reagents in aluminium-coated pouches is stable during accelerated storage and transport tests. When the limit of detection was assessed, we found that the cartridge-automated workflow consistently detected 10 CFU ml-1 of mycobacteria in spiked sputum samples. First tests with clinical samples showed a 100% specificity for non-TB specimens. In addition, Mycobacterium tuberculosis (MTB) was re-found in pre-characterized smear microscopy and culture positive sputum samples suggesting a high diagnostic sensitvity. In summary, the novel cartridge-automated workflow enables a flexible and sensitive TB diagnosis without the need to invest in specialized instrumentation.

Background Comprehensive and reliable drug susceptibility testing (DST) is urgently needed to provide adequate treatment regimens for patients with multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB). We determined whether next-generation sequencing (NGS) analysis of Mycobacterium tuberculosis complex isolates and genes implicated in drug resistance can guide the design of effective MDR/RR-TB treatment regimens. Methods NGS-based genomic DST predictions of M. tuberculosis complex isolates from MDR/RR-TB patients admitted to a TB reference center in Germany between 1 January 2015 and 30 April 2019 were compared with phenotypic DST results of mycobacteria growth indicator tubes (MGIT). Standardized treatment algorithms were applied to design individualized therapies based on either genomic or phenotypic DST results, and discrepancies were further evaluated by determination of minimal inhibitory drug concentrations (MICs) using Sensititre MYCOTBI and UKMYC microtiter plates. Results In 70 patients with MDR/RR-TB, agreement among 1048 pairwise comparisons of genomic and phenotypic DST was 86.3%; 76 (7.2%) results were discordant, and 68 (6.5%) could not be evaluated due to the presence of polymorphisms with yet unknown implications for drug resistance. Importantly, 549 of 561 (97.9%) predictions of drug susceptibility were phenotypically confirmed in MGIT, and 27 of 64 (42.2%) false-positive results were linked to previously described mutations mediating a low or moderate MIC increase. Virtually all drugs (99.0%) used in combination therapies that were inferred from genomic DST were confirmed to be susceptible by phenotypic DST. Conclusions NGS-based genomic DST can reliably guide the design of effective MDR/RR-TB treatment regimens.

Failure to rapidly identify drug-resistant tuberculosis (TB) increases the risk of patient mismanagement, the amplification of drug resistance, and ongoing transmission. We generated comparative analytical data for four automated assays for the detection of TB and multidrug-resistant TB (MDR-TB): Abbott RealTime MTB and MTB RIF/INH (Abbott), Hain Lifescience FluoroType MTBDR (Hain), BD Max MDR-TB (BD), and Roche cobas MTB and MTB-RIF/INH (Roche)., Failure to rapidly identify drug-resistant tuberculosis (TB) increases the risk of patient mismanagement, the amplification of drug resistance, and ongoing transmission. We generated comparative analytical data for four automated assays for the detection of TB and multidrug-resistant TB (MDR-TB): Abbott RealTime MTB and MTB RIF/INH (Abbott), Hain Lifescience FluoroType MTBDR (Hain), BD Max MDR-TB (BD), and Roche cobas MTB and MTB-RIF/INH (Roche). We included Xpert MTB/RIF (Xpert) and GenoType MTBDRplus as comparators for TB and drug resistance detection, respectively. We assessed analytical sensitivity for the detection of the Mycobacterium tuberculosis complex using inactivated strains (M. tuberculosis H37Rv and M. bovis) spiked into TB-negative sputa and computed the 95% limits of detection (LOD95). We assessed the accuracy of rifampicin and isoniazid resistance detection using well-characterized M. tuberculosis strains with high-confidence mutations accounting for >85% of first-line resistance mechanisms globally. For H37Rv and M. bovis, we measured LOD95 values of 3,781 and 2,926 (Xpert), 322 and 2,182 (Abbott), 826 and 4,301 (BD), 10,398 and 23,139 (Hain), and 2,416 and 2,136 (Roche) genomes/ml, respectively. Assays targeting multicopy genes or targets (Abbott, BD, and Roche) showed increased analytical sensitivity compared to Xpert. Quantification of the panel by quantitative real-time PCR prevents the determination of absolute values, and results reported here can be interpreted for comparison purposes only. All assays showed accuracy comparable to that of Genotype MTBDRplus for the detection of rifampicin and isoniazid resistance. The data from this analytical study suggest that the assays may have clinical performances similar to those of WHO-recommended molecular TB and MDR-TB assays.

Background Multidrug-resistant (MDR) Mycobacterium tuberculosis complex strains not detected by commercial molecular drug susceptibility testing (mDST) assays due to the RpoB I491F resistance mutation are threatening the control of MDR tuberculosis (MDR-TB) in Eswatini. Methods We investigate the evolution and spread of MDR strains in Eswatini with a focus on bedaquiline (BDQ) and clofazimine (CFZ) resistance using whole-genome sequencing in two collections ((1) national drug resistance survey, 2009–2010; (2) MDR strains from the Nhlangano region, 2014–2017). Results MDR strains in collection 1 had a high cluster rate (95%, 117/123 MDR strains) with 55% grouped into the two largest clusters (gCL3, n = 28; gCL10, n = 40). All gCL10 isolates, which likely emerged around 1993 (95% highest posterior density 1987–1998), carried the mutation RpoB I491F that is missed by commercial mDST assays. In addition, 21 (53%) gCL10 isolates shared a Rv0678 M146T mutation that correlated with elevated minimum inhibitory concentrations (MICs) to BDQ and CFZ compared to wild type isolates. gCL10 isolates with the Rv0678 M146T mutation were also detected in collection 2. Conclusion The high clustering rate suggests that transmission has been driving the MDR-TB epidemic in Eswatini for three decades. The presence of MDR strains in Eswatini that are not detected by commercial mDST assays and have elevated MICs to BDQ and CFZ potentially jeopardizes the successful implementation of new MDR-TB treatment guidelines. Measures to limit the spread of these outbreak isolates need to be implemented urgently.

Molecular tests to detect the presence of Mycobacterium tuberculosis and genetic polymorphisms in the rpoB gene conferring resistance to rifampicin (RMP) have become integral parts of tuberculosis diagnostics worldwide. These assays are often performed sequentially or in parallel to phenotypic drug susceptibility testing. Discordances between molecular and phenotypic tests invariably occur. Root causes range from pre-, post- and analytic errors to co-existence of non-tuberculous mycobacteria, silent mutations, mutations outside the 81 base-pair RMP resistance-determining region, non - canonical mutations conferring increased minimal inhibitory concentrations below the critical concentration in some phenotypic drug susceptibility tests, and heteroresistance. Resolving discordant results is challenging. This guide aims to assist both clinicians and microbiologists in interpreting discordances by providing a structured approach to manage further investigations. Case scenarios are discussed, including the likelihood of occurrence.

SETTING: Xpert® MTB/RIF is the most widely used molecular assay for rapid diagnosis of tuberculosis (TB). The number of polymerase chain reaction cycles after which detectable product is generated (cycle threshold value, CT) correlates with the bacillary burden. OBJECTIVE : To investigate the association between Xpert CT values and smear status through a systematic review and individual-level data meta-analysis. DESIGN: Studies on the association between CT values and smear status were included in a descriptive systematic review. Authors of studies including smear, culture and Xpert results were asked for individual-level data, and receiver operating characteristic curves were calculated. RESULTS : Of 918 citations, 10 were included in the descriptive systematic review. Fifteen data sets from studies potentially relevant for individual-level data meta-analysis provided individual-level data (7511 samples from 4447 patients); 1212 patients had positive Xpert results for at least one respiratory sample (1859 samples overall). ROC analysis revealed an area under the curve (AUC) of 0.85 (95%CI 0.82-0.87). Cut-off CT values of 27.7 and 31.8 yielded sensitivities of 85% (95%CI 83-87) and 95% (95%CI 94-96) and specificities of 67% (95%CI 66-77) and 35% (95%CI 30-41) for smear-positive samples. CONCLUSION: Xpert CT values and smear status were strongly associated. However, diagnostic accuracy at set cut-off CT values of 27.7 or 31.8 would not replace smear microscopy. How CT values compare with smear microscopy in predicting infectiousness remains to be seen. © 2017 The Union.

The Abbott RealTi m e MTB (RT MTB) assay is a new automated nucleic acid amplification test for the detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. In combination with the RealTi m e MTB INH/RIF (RT MTB INH/RIF) resistance assay, which can be applied to RT MTB-positive specimens as an add-on assay, the tests also indicate the genetic markers of resistance to isoniazid (INH) and rifampin (RIF). We aimed to evaluate the diagnostic sensitivity and specificity of RT MTB using different types of respiratory and extrapulmonary specimens and to compare performance characteristics directly with those of the FluoroType MTB assay. The resistance results obtained by RT MTB INH/RIF were compared to those from the GenoType MTBDR plus and from phenotypic drug susceptibility testing. A total of 715 clinical specimens were analyzed. Compared to culture, the overall sensitivity of RT MTB was 92.1%; the sensitivity rates for smear-positive and smear-negative samples were 100% and 76.2%, respectively. The sensitivities of smear-negative specimens were almost identical for respiratory (76.3%) and extrapulmonary (76%) specimens. Specificity rates were 100% and 95.8% for culture-negative specimens and those that grew nontuberculous mycobacteria, respectively. RT MTB INH/RIF was applied to 233 RT MTB-positive samples and identified resistance markers in 7.7% of samples. Agreement with phenotypic and genotypic drug susceptibility testing was 99.5%. In conclusion, RT MTB and RT MTB INH/RIF allow for the rapid and accurate diagnosis of tuberculosis (TB) in different types of specimens and reliably indicate resistance markers. The strengths of this system are the comparably high sensitivity with paucibacillary specimens, its ability to detect INH and RIF resistance, and its high-throughput capacities.

Multidrug-resistant tuberculosis (MDR-TB) is an emerging public health problem in Nepal. Despite the implementation of a successful TB control program in Nepal, notifications of MDR-TB are increasing, yet the reasons are unknown. The objective of this study was to understand the genetic diversity and epidemiological characteristics of MDR-Mycobacterium tuberculosis (MTB) isolates in Nepal. We isolated and genotyped 498 MDR-MTB isolates collected from April 2009 to March 2013 and analyzed the patients’ background information. Our results showed that the lineage 2 (Beijing family) was the most predominant lineage (n = 241; 48.4%), followed by lineage 3 (n = 153, 30.7%). Lineage 4 was the third most prevalent (n = 73, 14.5%) followed by lineage 1 (n = 32, 6.4%). The lineages were significantly associated with geographic region, ethnic group, age and sex of patients. The Beijing genotype was found to have an important role in transmitting MDR-TB in Nepal and was significantly associated with the eastern region, mongoloid ethnic group and younger age group. We conclude that early diagnosis and treatment including molecular-epidemiological surveillance of MDR-TB cases will help to control transmission of MDR-TB in Nepal.

OBJECTIVES The objective of this study was to develop standardized protocols for rapid delamanid drug susceptibility testing (DST) using the colorimetric resazurin microtitre assay (REMA) and semi-automated BACTEC™ MGIT™ 960 system (MGIT) by establishing breakpoints that accurately discriminate between susceptibility and resistance of Mycobacterium tuberculosis to delamanid. METHODS MICs of delamanid were determined by the MGIT, the REMA and the solid agar method for 19 pre-characterized strains. The MIC distribution of delamanid was then established for a panel of clinical strains never exposed to the drug and characterized by different geographical origins and susceptibility patterns. WGS was used to investigate genetic polymorphisms in five genes (ddn, fgd1, fbiA, fbiB and fbiC) involved in intracellular delamanid activation. RESULTS We demonstrated that the REMA and MGIT can both be used for the rapid and accurate determination of delamanid MIC, showing excellent concordance with the solid agar reference method, as well as high reproducibility and repeatability. We propose the tentative breakpoint of 0.125 mg/L for the REMA and MGIT, allowing reliable discrimination between M. tuberculosis susceptible and resistant to delamanid. Stop codon mutations in ddn (Trp-88 → STOP) and fbiA (Lys-250 → STOP) have only been observed in strains resistant to delamanid. CONCLUSIONS We established protocols for DST of delamanid in the MGIT and REMA, confirming their feasibility in routine TB diagnostics, utilizing the same discriminative concentration for both methods. Moreover, taking advantage of WGS analysis, we identified polymorphisms potentially associated with resistance in two genes involved in delamanid activation.

Non-tuberculous mycobacteria (NTM) include more than 160 ubiquitous, environmental, acid-fast-staining bacterial species, some of which may cause disease in humans. Chronic pulmonary infection is the most common clinical manifestation. Although patients suffering from chronic lung diseases are particularly susceptible to NTM pulmonary disease, many affected patients have no apparent risk factors. Host and pathogen factors leading to NTM pulmonary disease are not well understood and preventive therapies are lacking. NTM isolation and pulmonary disease are reported to rise in frequency in Europe as well as in other parts of the world. Differentiation between contamination, infection, and disease remains challenging. Treatment of NTM pulmonary disease is arduous, lengthy, and costly. Correlations between results of in vitro antibiotic susceptibility testing and clinical treatment outcomes are only evident for the Mycobacterium avium complex, M. kansasii, and some rapidly growing mycobacteria. We describe the epidemiology of NTM pulmonary disease as well as emerging NTM pathogens and their geographical distribution in non-cystic fibrosis patients in Europe. We also review recent innovations for the diagnosis of NTM pulmonary disease, summarize treatment recommendations, and identify future research priorities to improve the management of patients affected by NTM pulmonary disease.

International audience; We study a seasonally recurrent cyclone and related cloud phenomena observed on Mars at Ls ~ 120°, latitude ~ 60°N and longitude 90°W from images obtained with cameras in different spacecraft between 1995 and 2018. A remarkable double cyclone formed in 2012 and we present a detailed study of its dynamics using images from Mars Express and Mars Reconnaissance Orbiter obtained between June 6 and July 9. A double cyclone was also observed in 2006 and 2008. In other Martian Years the primary cyclone showed an annular cloud morphology with a large water ice cloud observed eastward of it. The cyclones have a size of ~ 600‐800 km with a cloud‐free core of a radius ~ 100‐300 km. Tangential velocities measured from cloud tracking in 2012 images are ~ 5‐20 ms‐1 at 10 km altitude and DC moved eastwards with a velocity of 4 ms‐ 1 during its lifetime of one month. The vortices grow in the morning hours, but with the increasing insolation as the sol progresses, a part of the clouds evaporate, the winds weaken and the vortices lose coherence. This phenomenon forms under high temperature gradients in a region with a large north‐south topographic slope and has been recurrent each Martian year between 1995 and 2018. We argue the interest of studying its changing properties each Martian year in order to explore their possible relationship to the state of the Martian atmosphere at Ls ~ 120°.

The law appears in legal texts and in typed legal situations. Textuality began to dominate with the reception of Roman law. The advancement of socially and legally used machines will decrease the importance of textuality and increase that of legal programming. Legal visualization fits this trend. The peculiarities of law form an object of visualization. Legal texts are in an abstract frame, which is not linguistically structured. Besides the types of legal situations, the pre-textual interdependencies of legal terms should be made visible. Situations can be governed by legal machines.