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Item does not contain fulltext BACKGROUND: Since 2013, over 100 cases of Mycobacterium chimaera prosthetic valve endocarditis and disseminated disease were notified in Europe and the USA, linked to contaminated heater-cooler units (HCUs) used during cardiac surgery. We did a molecular epidemiological investigation to establish the source of these patients' disease. METHODS: We included 24 M chimaera isolates from 21 cardiac surgery-related patients in Switzerland, Germany, the Netherlands, and the UK, 218 M chimaera isolates from various types of HCUs in hospitals, from LivaNova (formerly Sorin; London, UK) and Maquet (Rastatt, Germany) brand HCU production sites, and unrelated environmental sources and patients, as well as eight Mycobacterium intracellulare isolates. Isolates were analysed by next-generation whole-genome sequencing using Illumina and Pacific Biosciences technologies, and compared with published M chimaera genomes. FINDINGS: Phylogenetic analysis based on whole-genome sequencing of 250 isolates revealed two major M chimaera groups. Cardiac surgery-related patient isolates were all classified into group 1, in which all, except one, formed a distinct subgroup. This subgroup also comprised isolates from 11 cardiac surgery-related patients reported from the USA, most isolates from LivaNova HCUs, and one from their production site. Isolates from other HCUs and unrelated patients were more widely distributed in the phylogenetic tree. INTERPRETATION: HCU contamination with M chimaera at the LivaNova factory seems a likely source for cardiothoracic surgery-related severe M chimaera infections diagnosed in Switzerland, Germany, the Netherlands, the UK, the USA, and Australia. Protective measures and heightened clinician awareness are essential to guarantee patient safety. FUNDING: Partly funded by the EU Horizon 2020 programme, its FP7 programme, the German Center for Infection Research (DZIF), the Swiss National Science Foundation, the Swiss Federal Office of Public Health, and National Institute of Health Research Oxford Health Protection Research Units on Healthcare Associated Infection and Antimicrobial Resistance.

The Mycobacterium tuberculosis-harboring granuloma with a necrotic center surrounded by a fibrous capsule is the hallmark of tuberculosis (TB). For a successful treatment, antibiotics need to penetrate these complex structures to reach their bacterial targets. Hence, animal models reflecting the pulmonary pathology of TB patients are of particular importance to improve the preclinical validation of novel drug candidates. M. tuberculosis-infected interleukin-13-overexpressing (IL-13

Background In 2019, new therapeutic recommendations for multidrug-resistant (MDR-) and extensively drug-resistant (XDR) tuberculosis (TB) were published by the WHO, advocating the use of oral drugs and stepwise composition of antibiotic regimens. To date, the economic consequences of those recommendations in low incidence settings have not been evaluated. Objective To assess the costs of applying the new recommendations against a set of 86 MDR-TB/XDR-TB strains, each with individual phenotypic drug resistance patterns, identified in 2018/2019 by the German National Reference Center for Mycobacteria. Methods Hospitalization costs as covered by German statutory health insurance and the loss of productivity due to illness were calculated using the most recent 2018 statistical data. Costs due to combining five agents in the intensive phase and costs of outpatient monitoring were determined by Monte-Carlo simulation covering all treatment options over an 18-month period. Drug costs were compared to those arising under the approach recommended by the WHO in 2016. Results Hospitalization costs per MDR-TB patient were €30,152 and the mean costs of antimicrobials over a period of 18 months were €66,854 (range €20,671 to €187,444). Total treatment costs, including outpatient monitoring, were €73,551.56 per patient (range €30,114 to €145.878). In addition, we determined an average cost of €11,410.20 due to productivity loss over a period of 6 months sick leave. Despite a shortened minimum recommended treatment duration (18 versus 20 months), the estimated costs were 24.5% higher based on the 2019 recommendations as compared to the 2016 guideline version. Conclusion Higher costs for treating MDR-TB/XDR-TB in Germany are to be expected under the new WHO regimens. However, it must be determined whether treatment duration and costs associated with sick leave may be further reduced in the future through shorter hospital stays and earlier culture conversion.

ZusammenfassungBei 2 Hunden wurden im Rahmen der dermatologischen Untersuchung noduläre Hautläsionen im Kopf-/Ohrmuschelbereich nachgewiesen, die mittels histopathologischer, zytologischer und molekularbiologischer Untersuchungen als kanine leproide Granulome charakterisiert werden konnten. Die Erkrankung wird durch nichttuberkulöse, bisher nicht vollständig klassifizierte Mykobakterien verursacht und stellt eine im pazifischen Raum sowie in Nord- und Südamerika dokumentierte Hautläsion dar, die in Deutschland bisher nicht beschrieben wurde. Durch die in beiden Fällen erfolgte Kombination aus chirurgischer und medikamentöser Langzeittherapie (Rifampicin, Clarithromycin, Doxycyclin und lokal Clofazimin) konnten die Granulome entfernt bzw. zur Regression gebracht werden.

Tapirs are a taxonomic group with a high susceptibility to mycobacterial diseases. However, successful therapy has only been documented sporadically. Here treatment of mycobacteriosis diagnosed in three, one male and two female, lowland tapirs (Tapirus terrestris) in a zoo in Germany is reported. Two of the animals showed chronic mild respiratory signs, and conventional therapy did not improve the condition. Culture of broncho-alveolar lavage (BAL) samples was positive for Mycobacterium avium ssp. hominissuis. Upon airway endoscopy, bronchial edema and increased mucus production were visible. Initially, all three infected tapirs received oral antimycobacterial therapy consisting of 5 mg/kg body weight isoniazid, 10 mg/kg rifampicin, and 10 mg/kg clarithromycin q24h. Based on therapeutic drug level monitoring, the doses of rifampicin were adjusted to 12 and 15 mg/kg in the females and the male, respectively. The treatment with all three drugs was continued for 11 mon. Six months into treatment, the clinical condition resolved, and repeated BAL samples of all three tapirs tested negative for mycobacteria by culture. Here the approach for a treatment protocol with minimal side effects suitable to control infections with nontuberculous mycobacteria in lowland tapirs is reported.

Der Nachweis von Mycobacterium tuberculosis-Komplex-DNA durch PCR an formalinfixiertem Paraffinmaterial ist heutzutage integraler Bestandteil der molekularpathologischen Diagnostik. Wir beschreiben hier ein Verfahren, welches durch Anwendung von Mycobacterium szulgai als Positivkontrolle Kontaminationen detektiert und so zur Vermeidung falsch positiver Falle beitragt.

The clinical relevance of newly described nontuberculous mycobacteria is often unclear. We report a case of pulmonary infection caused by Mycobacterium hassiacum in an immunocompetent patient in Austria who had chronic obstructive pulmonary disease. Antimicrobial drug susceptibility testing showed low MICs for macrolides, aminoglycosides, fluoroquinolones, tetracyclines, imipenem, and linezolid.

ZusammenfassungBei einer Katze wurde eine atypische mykobakterielle Pannikulitis diagnostiziert. Mittels 16S-rRNA-Gen-Sequenzanalyse ließ sich Mycobacterium setense, ein grampositives stäbchenförmiges säurefestes Bakterium der Mycobacterium fortuitum-Gruppe nachweisen, das zuvor nie bei einem erkrankten Tier isoliert werden konnte. Initial wurden Resistenzen gegen Doxycyclin und Clarithromycin festgestellt und während der Behandlung mit Pradofloxacin entwickelte sich zudem eine Resistenz gegen Fluorchinolone, bedingt durch eine Mutation im Gyrasegen gyrA (S90W-Austausch). Trotz einer langfristigen antimikrobiellen Behandlung über 33 Monate wurde keine vollständige Heilung erzielt. Bei einer atypischen mykobakteriellen Pannikulitis ist die Speziesbestimmung mit Resistenztest die Grundlage für eine adäquate Antibiose.

Preexisting and newly emerging resistant pathogen subpopulations (heteroresistance) are potential risk factors for treatment failure of multi/extensively drug resistant (MDR/XDR) tuberculosis (TB). Intrapatient evolutionary dynamics of Mycobacterium tuberculosis complex (Mtbc) strains and their implications on treatment outcomes are still not completely understood. To elucidate how Mtbc strains escape therapy, we analyzed 13 serial isolates from a German patient by whole-genome sequencing. Sequencing data were compared with phenotypic drug susceptibility profiles and the patient’s collective 27-year treatment history to further elucidate factors fostering intrapatient resistance evolution. The patient endured five distinct TB episodes, ending in resistance to 16 drugs and a nearly untreatable XDR-TB infection. The first isolate obtained, during the patient’s 5th TB episode, presented fixed resistance mutations to 7 anti-TB drugs, including isoniazid, rifampin, streptomycin, pyrazinamide, prothionamide, para-aminosalicylic acid, and cycloserine-terizidone. Over the next 13 years, a dynamic evolution with coexisting, heterogeneous subpopulations was observed in 6 out of 13 sequential bacterial isolates. The emergence of drug-resistant subpopulations coincided with frequent changes in treatment regimens, which often included two or fewer active compounds. This evolutionary arms race between competing subpopulations ultimately resulted in the fixation of a single XDR variant. Our data demonstrate the complex intrapatient microevolution of Mtbc subpopulations during failing MDR/XDR-TB treatment. Designing effective treatment regimens based on rapid detection of (hetero) resistance is key to avoid resistance development and treatment failure.

Tapirs are a taxonomic group with a high susceptibility to mycobacterial diseases. However, successful therapy has only been documented sporadically. Here treatment of mycobacteriosis diagnosed in three, one male and two female, lowland tapirs (

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.

The detection of Mycobacterium tuberculosis complex DNA by PCR using formalin-fixed paraffin-embedded material has become an integral part of molecular-pathological diagnostics. We describe an approach that enables the detection of contamination by using Mycobacterium szulgai as a positive control, contributing to the reduction of false-positive results.Der Nachweis von Mycobacterium tuberculosis-Komplex-DNA durch PCR an formalinfixiertem Paraffinmaterial ist heutzutage integraler Bestandteil der molekularpathologischen Diagnostik. Wir beschreiben hier ein Verfahren, welches durch Anwendung von Mycobacterium szulgai als Positivkontrolle Kontaminationen detektiert und so zur Vermeidung falsch positiver Fälle beiträgt.

The cobas MTB and MTB-RIF/INH assays allow for detection of Mycobacterium tuberculosis complex (MTBC) nucleic acid and rifampicin (RIF) and isoniazid (INH) resistance-associated mutations in an automated, high-throughput workflow. In this study, we evaluated the performance of these assays, employing samples from settings of low and high tuberculosis (TB) burdens., The Roche cobas MTB and MTB-RIF/INH assays allow for detection of Mycobacterium tuberculosis complex (MTBC) nucleic acid and rifampicin (RIF) and isoniazid (INH) resistance-associated mutations in an automated, high-throughput workflow. In this study, we evaluated the performance of these assays, employing samples from settings of low and high tuberculosis (TB) burdens. A total of 325 frozen, leftover respiratory samples collected from treatment-naive patients with presumptive TB in Germany (n = 280) and presumptive RIF-resistant TB in Sierra Leone (n = 45) were used in this study. cobas MTB results for detection of MTBC DNA from N-acetyl-l-cysteine–sodium hydroxide (NALC–NaOH)-treated samples were compared to culture results. Predictions of RIF and INH resistance by the cobas MTB-RIF/INH assay were compared to a composite reference standard (phenotypic drug susceptibility testing and line probe assay). Whole-genome sequencing was used to resolve discordances. The overall sensitivity of cobas MTB for detection of MTBC DNA in culture-positive samples (n = 102) was 89.2% (95% confidence interval [CI], 81.7 to 93.9%). The specificity of cobas MTB was 98.6% (95% CI, 96.1 to 99.5%). Sensitivity and specificity for detection of RIF and INH resistance were 88.4% (95% CI, 75.5 to 94.9%) and 97.6% (95% CI, 87.4 to 99.6%) and 76.6% (95% CI, 62.8 to 86.4%) and 100.0% (95% CI, 90.8 to 100.0%), respectively. Discordant results for RIF and INH resistance were mainly due to uncommon mutations in samples from Sierra Leone that were not covered by the cobas MTB-RIF/INH assay. In conclusion, cobas MTB and MTB-RIF/INH assays provide accurate detection of MTBC DNA and resistance-associated mutations in respiratory samples. The influence of regional variations in the prevalence of resistance-conferring mutations requires further investigation.

Tapirs seem particularly susceptible to mycobacterial infections, especially to tuberculosis caused by M. tuberculosis or M. bovis. In this case series, we report an infection with the non-tuberculous mycobacteria (NTM) species M. avium ssp. hominissuis (MAH) in a group of four (2.2) captive lowland tapirs (Tapirus terrestris). Two female tapirs showed mild respiratory signs such as coughing and mucous sputum production for several years, one juvenile male tapir had to be euthanized due to severe dyspnoea, and the adult male only showed mild respiratory signs in 2010. Post-mortem histopathology of the euthanized animal revealed a chronic bronchopneumonia, and MAH was detected via culture. Subsequently, the three remaining tapirs were tested further: serologically, the tapirs had high antibody titres against M. avium, but they showed no reaction in the comparative skin test (TST). At several time points, the animals were tested for the presence of mycobacteria in different sample matrices including sputum samples, pooled faecal samples as well as swabs from the tapir enclosure to identify potential environmental niches of the pathogen. Moreover, animals were directly sampled using nasal swabs, endoscopic broncho-alveolar (BAL) and gastric lavages. MAH was detected by culture in the sputum samples, in the BAL of the breeding pair, as well as in the swimming pool water and walls, and in swabs taken from the tapir's sleeping beds. We conclude that the TST is not a useful diagnostic tool to detect MAC infections in tapirs, whereas antibody ELISA and culture from BAL appear more sensitive.

Using cytology, histopathology, and DNA sequencing the diagnosis of canine leproid granuloma (CLG) was made in 2 dogs. The dogs were presented with nodular skin lesions on the head and pinnae. CLG is caused by nontuberculous mycobacteria that have not yet been finally classified. To date, this disease has been reported in Australia, New Zealand as well as North and South America, however no case reports have been published in Germany until now. In both cases, a combination of surgery and long-term drug administration (rifampicin, clarithromycin, doxycyclin and local application of clofazimin) was chosen and successfully eliminated the granulomas.Bei 2 Hunden wurden im Rahmen der dermatologischen Untersuchung noduläre Hautläsionen im Kopf-/Ohrmuschelbereich nachgewiesen, die mittels histopathologischer, zytologischer und molekularbiologischer Untersuchungen als kanine leproide Granulome charakterisiert werden konnten. Die Erkrankung wird durch nichttuberkulöse, bisher nicht vollständig klassifizierte Mykobakterien verursacht und stellt eine im pazifischen Raum sowie in Nord- und Südamerika dokumentierte Hautläsion dar, die in Deutschland bisher nicht beschrieben wurde. Durch die in beiden Fällen erfolgte Kombination aus chirurgischer und medikamentöser Langzeittherapie (Rifampicin, Clarithromycin, Doxycyclin und lokal Clofazimin) konnten die Granulome entfernt bzw. zur Regression gebracht werden.

Background Tuberculosis (TB), especially drug-resistant TB, is a global public health problem. This study aimed to validate a new molecular diagnostic test, the FluoroType® MTBDR. Method Samples underwent routine diagnostic procedures (fluorescence microscopy, culture, species differentiation and phenotypic drug susceptibility testing). Left over samples stored at −20° underwent DNA extraction using the Fluorolyse® kit, followed by FluoroType® MTBDR and Genotype MTBDRplus testing. Results A total of 350 respiratory and 59 lymph node samples were included in the study; 71 respiratory and 16 lymph node samples were culture positive for M. tuberculosis complex (MTBC). The sensitivity of the FluoroType® MTBDR to detect MTBC DNA was 91.4% (95%CI 82.3–96.8%), 68.4% (95%CI 43.4–87.4%) and 62.5%, (95%CI 35.4–84.8%) for respiratory, smear negative respiratory and lymph node samples respectively. The correlating sensitivities of the GenoType MTBDRplus were 85.9% (95%CI 75.6–93.0%), 52.6% (95%CI 28.9–75.6%) and 56.3% (29.9–80.2). Sensitivity of the FluoroType® MTBDR to detect RMP and INH resistance for respiratory samples was 96.5% (95%CI 82.2–99.9) and 70% (95%CI 45.7–88.1), respectively. The GenoType MTBDRplus revealed sensitivities of 97.1% (95% 85.1–99.9) 70.6% (95%CI 52.5–84.9) for detection of RMP and INH resistance. Indeterminate results were 13/64 (20.3%), 23/64 (35.9%) and 16/64 (25.0%) for rpoB, katG and inhA using the FluoroType® MTBDR. Conclusion The FluoroType® MTBDR has a high sensitivity to detect MTBC DNA. However, the high proportion of indeterminate results across all three genes needs to be addressed.

Deutschland ist ein Niedriginzidenzland fur Tuberkulose. Gerade weil die Erkrankunge hier selten ist, muss sie bei der Anamnese als Differenzialdiagnose prasent sein. Fur die Diagnostik sind bildgebende Verfahren sowie mikro- und molekularbiologische Tests notig. Die Behandlung bei Tuberkulose ist langwierig, eine Standardtherapie dauert mindestens sechs Monate. Sind die Erreger gegen Standardmedikamente resistent, kann es deutlich langer dauern. Dann sind individuelle Losungen gefragt.

National Mycobacterium Reference Laboratory, Borstel, Germany. : To evaluate the effectiveness of OMNIgene®•SPUTUM (OM-S) reagent in comparison with a method using N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) with regard to mycobacterial recovery and contamination of broth and solid cultures. : Sputum samples from patients with tuberculosis and other respiratory diseases underwent decontamination with NALC-NaOH-based (MycoDDR™) or OM-S reagent. The decontamination procedure was assigned by block randomisation. Samples were inoculated on Lowenstein-Jensen, Stonebrink and MGIT™ (Mycobacterial Growth Indicator Tubes). Mycobacterial recovery from samples spiked with Mycobacterium tuberculosis following decontamination was determined. : Eighty-five samples were randomised to NALC-NaOH and 84 to OM-S reagent. Mycobacterial recovery was significantly lower for samples processed with OM-S reagent compared with the NALC-NaOH method across all media types. Culture contamination was lower with NALC-NaOH reagent on solid media (9.4-12.9% vs. 28.6-29.8%). Growth was not observed in MGIT among samples spiked with 10 600-16 800 colony-forming units of M. tuberculosis following decontamination with OM-S reagent. : Low mycobacterial recovery, especially in MGIT, observed in the present study suggests that OM-S reagent might not be compatible with the MGIT system. More extensive field evaluations of the OM-S reagent are warranted to demonstrate a significant benefit over currently used methods.

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.

A 9-month old pet ferret was presented to a veterinarian with symptoms of weight loss, apathy, and hyporexia. Explorative laparotomy identified a firm mass of approximately 2 × 2 × 2 cm in size in the mesentery of the jejunum. Because of the poor general condition and the unfavorable prognosis, the ferret was euthanized during surgery. The mass was resected in total and submitted to histological examination which revealed a granulomatous and necrotizing lymphadenitis. Acid fast bacteria were detected by Fite-Faraco staining leading to the suspicion of an infection with Mycobacteria sp. PCR confirmed presence of DNA of members of the Mycobacterium tuberculosis complex, subsequently specified as M. bovis. The detected spoligotype SB2548 was described for the first time. Ferrets are presented to veterinarians with increasing frequency because of their growing popularity as pet animals. Since these animals are highly susceptible to mycobacterial infections, mycobacteriosis and especially zoonotic relevant tuberculosis should be considered as differential diagnosis.

In many settings, the ongoing coronavirus disease (COVID-19) pandemic coincides with other major public health threats, in particular tuberculosis. Using tuberculosis (TB) molecular diagnostic infrastructure, which has substantially expanded worldwide in recent years, for COVID-19 case-finding might be warranted. We analyze the potential of using TB diagnostic and research infrastructures for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. We focused on quality control by adapting the 12 Quality System Essentials framework to the COVID-19 and TB context. We conclude that diagnostic infrastructures for TB can in principle be leveraged to scale-up SARS-CoV-2 testing, in particular in resource-poor settings. TB research infrastructures also can support sequencing of SARS-CoV-2 to study virus evolution and diversity globally. However, fundamental principles of quality management must be followed for both TB and SARS-CoV-2 testing to ensure valid results and to minimize biosafety hazards, and the continuity of TB diagnostic services must be guaranteed at all times.

Wollamides are cyclic hexapeptides, recently isolated from an Australian soil Streptomyces isolate, that exhibit promising in vitro antimycobacterial activity against Mycobacterium bovis Bacille Calmette Guérin without displaying cytotoxicity against a panel of mammalian cells. Here, we report the synthesis and antimycobacterial activity of 36 new synthetic wollamides, collated with all known synthetic and natural wollamides, to reveal structure characteristics responsible for in vitro growth-inhibitory activity against Mycobacterium tuberculosis (H37Rv, H37Ra, CDC1551, HN878, and HN353). The most potent antimycobacterial wollamides were those where residue VI d-Orn (wollamide B) was replaced by d-Arg (wollamide B1) or d-Lys (wollamide B2), with all activity being lost when residue VI was replaced by Gly, l-Arg, or l-Lys (wollamide B3). Substitution of other amino acid residues mainly reduced or ablated antimycobacterial activity. Significantly, whereas wollamide B2 was the most potent in restricting M. tuberculosis in vitro, wollamide B1 restricted M. tuberculosis intracellular burden in infected macrophages. Wollamide B1 synergized with pretomanid (PA-824) in inhibiting M. tuberculosis in vitro growth but did not antagonize prominent first- and second-line tuberculosis antibiotics. Furthermore, wollamide B1 exerted bactericidal activity against nonreplicating M. tuberculosis and impaired growth of multidrug- and extensively drug-resistant clinical isolates. In vivo pharmacokinetic profiles for wollamide B1 in rats and mice encourage further optimization of the wollamide pharmacophore for in vivo bioavailability. Collectively, these observations highlight the potential of the wollamide antimycobacterial pharmacophore.

Less than 30% of multidrug-resistant tuberculosis (MDR-TB) patients are currently diagnosed, due to laboratory constraints. Molecular diagnostics enable rapid and simplified diagnosis. Newer-version line probe assays have not been evaluated against the WHO-endorsed Hain GenoType MTBDR plus (referred to as Hain version 1 [V1]) for the rapid detection of rifampin (RIF) and isoniazid (INH) resistance. A two-phase noninferiority study was conducted in two supranational reference laboratories to allow head-to-head comparisons of two new tests, Hain Genotype MTBDR plus version 2 (referred to as Hain version 2 [V2]) and Nipro NTM+MDRTB detection kit 2 (referred to as Nipro), to Hain V1. In phase 1, the results for 379 test strains were compared to a composite reference standard that used phenotypic drug susceptibility testing (DST) and targeted sequencing. In phase 2, the results for 644 sputum samples were compared to a phenotypic DST reference standard alone. Using a challenging set of strains in phase 1, the values for sensitivity and specificity for Hain V1, Hain V2, and Nipro, respectively, were 90.3%/98.5%, 90.3%/98.5%, and 92.0%/98.5% for RIF resistance detection and 89.1%/99.4%, 89.1%/99.4%, and 89.6%/100.0% for INH resistance detection. Testing of sputa in phase 2 yielded values for sensitivity and specificity of 97.1%/97.1%, 98.2%/97.8%, and 96.5%/97.5% for RIF and 94.4%/96.4%, 95.4%/98.8%, and 94.9%/97.6% for INH. Overall, the rates of indeterminate results were low, but there was a higher rate of indeterminate results with Nipro than with Hain V1 and V2 in samples with low smear grades. Noninferiority of Hain V2 and Nipro to Hain V1 was demonstrated for RIF and INH resistance detection in isolates and sputum specimens. These results serve as evidence for WHO policy recommendations on the use of line probe assays, including the Hain V2 and Nipro assays, for MDR-TB detection.

Phenotypic drug susceptibility testing (DST) for the two first-line tuberculosis drugs ethambutol and pyrazinamide is known to yield unreliable and inaccurate results. In this prospective study, we propose a diagnostic algorithm combining phenotypic DST with Sanger sequencing to inform clinical decision-making for drug-resistant Mycobacterium tuberculosis complex isolates., Phenotypic drug susceptibility testing (DST) for the two first-line tuberculosis drugs ethambutol and pyrazinamide is known to yield unreliable and inaccurate results. In this prospective study, we propose a diagnostic algorithm combining phenotypic DST with Sanger sequencing to inform clinical decision-making for drug-resistant Mycobacterium tuberculosis complex isolates. Sequencing results were validated using whole-genome sequencing (WGS) of the isolates. Resistance-conferring mutations obtained by pncA sequencing correlated well with phenotypic DST results for pyrazinamide. Phenotypic resistance to ethambutol was only partly explained by mutations in the embB 306 codon. Additional resistance-conferring mutations were found in the embB gene at codons 354, 406, and 497. In several isolates that tested ethambutol susceptibility by phenotypic DST, well-known resistance-conferring embB mutations were determined. Thus, targeted Sanger sequencing beyond the embB 306 codon or WGS together with phenotypic DST should be employed to ensure reliable ethambutol drug susceptibility testing, as a basis for the rational design of multidrug-resistant tuberculosis regimens with or without ethambutol.

We would like to thank Dr. Navisha Dookie and colleagues for their comment and for sharing their view on genotypic drug-susceptibility testing (DST) to predict phenotypic drug-susceptibility for the management of patients with drug-resistant tuberculosis (1, 2). We agree that DST should ideally be conducted for all drugs in a regimen, particularly given the severe adverse effects of second-line anti-tuberculosis drugs. Yet, even whole-genome sequencing in not a one-size-fits-all solution. Instead, the goal has to be to optimally combine genotypic and phenotypic assays in a diagnostic algorithm that capitalises on the strengths of both approaches whilst including appropriate reflex and confirmatory testing to minimize the impact of their respective limitations, including systematic errors (3).

Tuberculosis (TB), especially drug-resistant TB, is a global public health problem. This study aimed to validate a new molecular diagnostic test, the FluoroTypeSamples underwent routine diagnostic procedures (fluorescence microscopy, culture, species differentiation and phenotypic drug susceptibility testing). Left over samples stored at -20° underwent DNA extraction using the FluorolyseA total of 350 respiratory and 59 lymph node samples were included in the study; 71 respiratory and 16 lymph node samples were culture positive for M. tuberculosis complex (MTBC). The sensitivity of the FluoroTypeThe FluoroType

Objectives To determine MIC distributions for Mycobacterium chimaera, Mycobacterium intracellulare, Mycobacterium colombiense and Mycobacterium avium, and to derive tentative epidemiological cut-off (ECOFF) values. Methods A total of 683 bacterial isolates (M. chimaera, n = 203; M. intracellulare, n = 77; M. colombiense, n = 68; M. avium, n = 335) from 627 patients were tested by broth microdilution according to CLSI protocol M24-A2 on Sensititre RAPMYCOI plates. MICs were interpreted based on CLSI breakpoints for clarithromycin, and tentative breakpoints for amikacin, moxifloxacin and linezolid. Tentative ECOFFs were determined by visual approximation and the ECOFFinder algorithm. Results Modal MIC, MIC50 and MIC90 values were within ± one dilution step from the respective aggregated data set for 47/48 (97.9%), 48/48 (100%) and 48/48 (100%) species–drug combinations. Clarithromycin wild-type populations were mostly classified as susceptible (MIC90 4–8 mg/L; S ≤8 mg/L). Rifabutin MICs were lower than those of rifampicin. Tentative moxifloxacin, linezolid and amikacin breakpoints split wild-type populations. No ECOFFs could be set for rifampicin, ethambutol, ciprofloxacin, isoniazid, trimethoprim/sulfamethoxazole and doxycycline because of truncation of MIC distributions. Agreement between the visually determined and the modelled 97.5% ECOFFs was 90.9%. All 99.0% ECOFFs were one titre step higher than by visual approximation. Conclusions Drug susceptibility patterns of M. chimaera are comparable to those of closely related species. Except for clarithromycin, breakpoints for Mycobacterium avium-intracellulare complex should be re-evaluated. Statistical determination of the 99.0% ECOFF may be superior to visual approximation.

The emergence of drug-resistant strains of Mycobacterium tuberculosis is a challenge to global tuberculosis (TB) control. Although culture-based methods have been regarded as the gold standard for drug susceptibility testing (DST), molecular methods provide rapid information on mutations in the M. tuberculosis genome associated with resistance to anti-tuberculosis drugs. We ascertained consensus on the use of the results of molecular DST for clinical treatment decisions in TB patients. This document has been developed by TBNET and RESIST-TB groups to reach a consensus about reporting standards in the clinical use of molecular DST results. Review of the available literature and the search for evidence included hand-searching journals and searching electronic databases. The panel identified single nucleotide mutations in genomic regions of M. tuberculosis coding for katG, inhA, rpoB, embB, rrs, rpsL and gyrA that are likely related to drug resistance in vivo. Identification of any of these mutations in clinical isolates of M. tuberculosis has implications for the management of TB patients, pending the results of in vitro DST. However, false-positive and false-negative results in detecting resistance-associated mutations in drugs for which there is poor or unproven correlation between phenotypic and clinical drug resistance complicate the interpretation. Reports of molecular DST results should therefore include specific information on the mutations identified and provide guidance for clinicians on interpretation and on the choice of the appropriate initial drug regimen.

Our objective was to assess the cost–benefit of enhancing or replacing the conventional sputum smear with the real-time PCR Xpert MTB/RIF method in the inpatient diagnostic schema for tuberculosis (TB).Recent data from published per-case cost studies for TB/multidrug-resistant (MDR)-TB and from comparative analyses of sputum microscopy, mycobacterial culture, Xpert MTB/RIF and drug susceptibility testing, performed at the German National Reference Center for Mycobacteria, were used. Potential cost savings of Xpert MTB/RIF, based on test accuracy and multiple cost drivers, were calculated for diagnosing TB/MDR-TB suspects from the hospital perspective.Implementing Xpert MTB/RIF as an add-on in smear-positive and smear-negative TB suspects saves on average €48.72 and €503, respectively, per admitted patient as compared with the conventional approach. In smear-positive and smear-negative MDR-TB suspects, cost savings amount to €189.56 and €515.25 per person, respectively. Full replacement of microscopy by Xpert MTB/RIF saves €449.98. In probabilistic Monte-Carlo simulation, adding Xpert MTB/RIF is less costly in 46.4% and 76.2% of smear-positive TB and MDR-TB suspects, respectively, but 100% less expensive in all smear-negative suspects. Full replacement by Xpert MTB/RIF is also consistently cost-saving.Using Xpert MTB/RIF as an add-on to and even as a replacement for sputum smear examination may significantly reduce expenditures in TB suspects.

Resistance to fluoroquinolones (FLQ) and second-line injectable drugs (SLID) is steadily increasing, especially in eastern European countries, posing a serious threat to effective tuberculosis (TB) infection control and adequate patient management. The availability of rapid molecular tests for the detection of extensively drug-resistant TB (XDR-TB) is critical in areas with high rates of multidrug-resistant TB (MDR-TB) and XDR-TB and limited conventional drug susceptibility testing (DST) capacity. We conducted a multicenter study to evaluate the performance of the new version (v2.0) of the Genotype MTBDR sl assay compared to phenotypic DST and sequencing on a panel of 228 Mycobacterium tuberculosis isolates and 231 smear-positive clinical specimens. The inclusion of probes for the detection of mutations in the e is promoter region in the MTBDR sl v2.0 test resulted in a higher sensitivity for detection of kanamycin resistance for both direct and indirect testing (96% and 95.4%, respectively) than that seen with the original version of the assay, whereas the test sensitivities for detection of FLQ resistance remained unchanged (93% and 83.6% for direct and indirect testing, respectively). Moreover, MTBDR sl v2.0 showed better performance characteristics than v1.0 for the detection of XDR-TB, with high specificity and sensitivities of 81.8% and 80.4% for direct and indirect testing, respectively. MTBDR sl v2.0 thus represents a reliable test for the rapid detection of resistance to second-line drugs and a useful screening tool to guide the initiation of appropriate MDR-TB treatment.

For Mycobacterium tuberculosis complex (MTBC), the rapid and accurate diagnosis of drug resistance is crucial to ensure early initiation of appropriate therapy. Recently, a new molecular diagnostic test, the FluoroType MTBDR, aimed at detecting rifampin and isoniazid resistance has become available. This study aimed to evaluate the FluoroType MTBDR in comparison to phenotypic drug susceptibility testing (DST) using M. tuberculosis complex isolates. MTBC isolates underwent phenotypic DST and were tested using the FluoroType MTBDR and Genotype MTBDRplus. Sanger sequencing of the key regions of rpoB , katG , inhA , and aphC was performed for isolates with discordant phenotypic and molecular results. Furthermore, isolates with specific wild-type bands missing in the Genotype MTBDRplus, indicating the presence of a mutation, were investigated by Sanger sequencing. Specificity and sensitivity, defined as the proportions of isolates correctly determined as susceptible and resistant by the FluoroType MTBDR compared to phenotypic DST, were calculated. A total of 180 culture isolates were included; phenotypic DST showed 85 isolates susceptible to isoniazid and rifampin, 7 with isoniazid monoresistance, 7 with rifampin monoresistance, and 81 with multidrug resistance. The specificity of the FluoroType MTBDR was 100% (95% confidence interval [CI], 96.0 to 100%) for both rifampin and isoniazid. The sensitivity was 91.7% (95% CI, 83.6 to 96.6%) for isoniazid and 98.9% (95% CI, 93.8 to 100.0%) for rifampin. The FluoroType MTBDR has a high sensitivity and specificity for the detection of rifampin and isoniazid resistance when using culture isolates.

Background. In Sudan, tuberculosis diagnosis largely relies on clinical symptoms and smear microscopy as in many other low- and middle-income countries. The aim of this study was to investigate the positive predictive value of a positive sputum smear in patients investigated for pulmonary tuberculosis in Eastern Sudan. Methods. Two sputum samples from patients presenting with symptoms suggestive of tuberculosis were investigated using direct Ziehl-Neelsen (ZN) staining and light microscopy between June to October 2014 and January to July 2016. If one of the samples was smear positive, both samples were pooled, stored at −20°C, and sent to the National Reference Laboratory (NRL), Germany. Following decontamination, samples underwent repeat microscopy and culture. Culture negative/contaminated samples were investigated using polymerase chain reaction (PCR). Results. A total of 383 samples were investigated. Repeat microscopy categorized 123 (32.1%) as negative, among which 31 were culture positive. This increased to 80 when PCR and culture results were considered together. A total of 196 samples were culture positive, of which 171 (87.3%), 14 (7.1%), and 11 (5.6%) were M. tuberculosis, M. intracellulare, and mixed species. Overall, 15.6% (57/365) of the samples had no evidence of M. tuberculosis, resulting in a positive predictive value of 84.4%. Conclusions. There was a discordance between the results of smear microscopy performed at local laboratories in the Sudan and at the NRL, Germany; besides, a considerable number of samples had no evidence of M. tuberculosis. Improved quality control for smear microscopy and more specific diagnostics are crucial to avoid possible overtreatment.

Simultaneous use of genotypic and phenotypic diagnostic tools for detection of rifampicin (RIF) susceptibility may yield discrepant results.To measure the discordance between the RIF-susceptibility results by Xpert MTB/RIF and Mycobacterium Growth Indicator Tube (MGIT), to evaluate if application of both tests to the same sample affects the discrepancy, and to evaluate treatment outcome in patients with the discordant strains.Sputa from patients with tuberculosis managed in the penitentiary system of Azerbaijan during 2011-2015 were examined for RIF susceptibility using Xpert MTB/RIF and MGIT. Strains with discrepant results were sequenced.Of 532 patients included, 6.2% had discordant RIF-susceptibility results. No significant association of the discordant RIF-susceptibility results with application of both tests on one sample versus sequential samples was found. L511P mutation accounted significantly (p = 0.006) for the discrepancy among those RIF resistant on Xpert MTB/RIF, but sensitive on MGIT. No significant association was identified between the outcomes of treatment with the first- or second-line drugs and the presence of any mutation.The Xpert MTB/RIF and MGIT testing may be used in sequential sputum samples without increase in the RIF-susceptibility discordance rate. L511P mutation significantly accounts for discordant RIF-susceptibility results, but its clinical relevance may be low.