OBJECTIVES:Mycobacterium haemophilum is a fastidious nontuberculous mycobacterium that causes severe infections in immunocompromised patients. Due to its complex growth requirements, its detection and antimicrobial susceptibility testing (AST) remain challenging. This study aimed to compare the performance of a modified commercial broth microdilution method (Sensititre™ Myco susceptibility plates with added haemin) with the standard Clinical and Laboratory Standards Institute (CLSI) agar disk elution method for AST of M. haemophilum. METHODS:We conducted 20 comparative AST tests on M. haemophilum ATCC 29548 and 10 clinical isolates, using the CLSI agar disk elution method as the reference. MICs were determined using both methods, and categorical concordance was assessed for multiple antibiotics. RESULTS:The overall concordance between methods was 81.4%. High agreement was observed for clarithromycin, linezolid, trimethoprim-sulfamethoxazole and rifampicin (90%-100%), while lower concordance was noted for amikacin (40%), ciprofloxacin (75%) and doxycycline (65%). Sensititre™ microdilution plates demonstrated superior standardization, ease of use and reduced contamination risks compared to the agar disk method. CONCLUSIONS:The modified Sensititre™ microdilution method is a reliable alternative to the agar disk elution method for AST of M. haemophilum. It provides precise MIC values and is better suited for routine clinical use due to improved reproducibility and efficiency.

Mycobacterium tuberculosis is well adapted to survive and persist in the infected host, escaping the host's immune response. Since polyamines such as spermine, which are synthesized by infected macrophages, are able to inhibit the growth of M. tuberculosis, the pathogen needs strategies to cope with these toxic metabolites. The actinomycete Streptomyces coelicolor, a close relative of M. tuberculosis, makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in M. tuberculosis. In the current study, we demonstrated that M. tuberculosis growth was inhibited by the polyamine spermine. Using in vitro enzymatic assays we determined that GlnA3(Mt) (Rv1878) possesses genuine gamma-glutamylspermine synthetase catalytic activity. We further showed that purified His-Strep-GlnA3(Mt), as well as native GlnA3(Mt), prefer spermine as a substrate over putrescine, cadaverine, spermidine, or other monoamines and amino acids, suggesting that GlnA3(Mt) may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the glnA3 gene in M. tuberculosis did not result in growth inhibition or enhanced sensitivity of M. tuberculosis in the presence of high spermine concentrations. Gene expression analysis of spermine-treated M. tuberculosis revealed no difference in the level of glnA3(Mt) expression relative to untreated cells, whereas a gene encoding a previously characterized efflux pump (Mmr; rv3065) was significantly upregulated. This suggests that bacterial survival under elevated spermine concentrations can not only be achieved by detoxification of spermine itself but also by mechanisms resulting in decreased spermine levels in the bacteria. IMPORTANCE Upon Mycobacterium tuberculosis infection macrophages synthesize the polyamine spermine, which at elevated concentrations is toxic for M. tuberculosis. Based on our investigations of spermine resistance in the closely related actinomycete Streptomyces coelicolor, we hypothesized that the glutamylspermine synthetase GlnA3 may be responsible for the resistance of M. tuberculosis against toxic spermine. Here we show that GlnA3(Mt) can indeed covalently modify spermine via glutamylation. However, GlnA3(Mt) is probably not the only resistance mechanism since a glnA3 null mutant of M. tuberculosis can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. Thus a combination of GlnA3(Mt) and specific efflux pumps acting as putative spermine transporters may constitute an active spermine-detoxification system in M. tuberculosis.

BACKGROUND:Recently, face mask sampling (FMS) confirmed detection of Mycobacterium tuberculosis DNA from exhaled breath in adults with tuberculosis. To date, no study has evaluated the use of FMS to detect pulmonary tuberculosis in children. We developed a method for FMS of M. tuberculosis-specific DNA in children and performed a clinical exploration to assess feasibility in children. METHODS:Face masks were spiked, analyzed on GeneXpert-Ultra, quantitative polymerase chain reaction, and targeted next-generation sequencing. Children with pulmonary tuberculosis were asked to wear 3 modified FFP2 masks for 30 minutes as part of an exploratory clinical study. RESULTS:Experiments with H37Ra M. tuberculosis strain showed a limit of 95% detection of 3.75 colony-forming units (95% confidence interval, 4.85-3.11) on GeneXpert-Ultra. Ten children with pulmonary tuberculosis participated in the clinical study. M. tuberculosis-specific DNA was detected on none of the face masks. CONCLUSIONS:Pediatric FMS has a low limit of detection for M. tuberculosis-specific DNA in vitro. However, M. tuberculosis DNA was not detected in any of 30 masks worn by children with pulmonary tuberculosis. This suggests that FMS in this form may not be more effective for detecting M. tuberculosis in children with tuberculosis than existing methods.

Background Mycobacterium chelonae is a rare cause of infective endocarditis that is difficult to diagnose and treat. After we found M chelonae in a series of patients, we aimed to investigate its role in cardiovascular prosthesis dysfunction and contamination of bioprostheses as a possible cause of infection. Methods In this collaborative microbiological study, we report on nine patients treated in three cardiovascular surgical departments in Germany, who were found to have M chelonae infection after receiving BioIntegral bioprostheses. We performed fluorescence in-situ hybridisation (FISH) combined with broad-range 16S rRNA gene amplification and sequencing (FISHseq) on samples of native cardiovascular tissue and explanted bioprosthetic material, as well as on 12 unused BioIntegral prostheses. We confirmed FISHseq findings with histological examination by staining for acid-fast bacilli, and M chelonae was differentiated from M abscessus by molecular techniques. Findings Between Dec 1, 2020, and Feb 28, 2022, we identified M chelonae in BioIntegral bioprostheses from three initial patients treated in Berlin that were explanted following dysfunction or suspected endocarditis, visualising morphologically intact FISH-positive mycobacteria. Despite negative mycobacterial culture, we also detected M chelonae in all 12 unused BioIntegral prostheses. The competent authorities in the EU prompted an alert, leading to the identification of six additional patients between March 1, 2022, and July 31, 2023. To find other cases of M chelonae endocarditis, we reviewed the FISHseq results of 1237 cardiovascular samples that were analysed between Jan 1, 2015, and Aug 31, 2022, including 295 samples from 228 bioprostheses supplied by other manufacturers. M chelonae was only detected in six of 41 patients who had received BioIntegral products. Interpretation Bioprostheses manufactured by BioIntegral Surgical might be colonised by M chelonae, which can lead to implant dysfunction. These infections are likely to be missed by conventional routine diagnostics and should be considered in patients with BioIntegral implants and suspected infection or dysfunction. Cases should be reported to public health and regulatory authorities. Routine safety testing of bioprostheses during manufacture should be reconsidered. Funding German Federal Ministry of Education and Research.

Objectives: We evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a secondstep real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures. Methods: FTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing. Results: Of the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and speci ficity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and speci ficities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and speci ficity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively. Discussion: FTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes. Erik Svensson, Clin Microbiol Infect 2024;30:1055 (c) 2024 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

ABSTRACT We assessed the performance of a novel real-time PCR-based transrenal DNA (trDNA) assay for the specific detection of Mycobacterium tuberculosis as a candidate marker of the early anti-tuberculosis therapy response. The study was performed on 288 urine samples from 72 tuberculosis patients collected at baseline and days 3, 7, and 14 of treatment with amoxicillin-clavulanic acid alone or in combination with meropenem, ertapenem, optimized-dose rifampicin, or standard treatment control in South Africa. trDNA was detected in one-third of the samples. The highest proportion of positive PCR results (cycle threshold < 36) was observed on days 3 and 7, reflecting the point in time when maximum bacterial killing and disintegration are expected. When analyzed by study arms, the trend was observed in groups treated with active antibiotics affecting cell wall integrity (meropenem, control) but not in inactive drugs (ertapenem, amoxicillin/clavulanic acid alone) or active drugs not affecting the cell wall (rifampicin). Overall, however, the trDNA assay did not correlate well with sputum culture-based decline of viable bacteria. This is possibly due to trDNA reflecting the killing of both culturable and non-culturable bacteria and should be explored further. IMPORTANCE This study presents the results of the evaluation of a novel method for the detection of Mycobacterium tuberculosis, the causative agent of tuberculosis, in urine. Detecting parts of the mycobacteria in urine is of particular interest as it allows us to use a sample that is easy to obtain and that does not require uncomfortable procedures or safety precautions like obtaining sputum for culture, which is the most commonly used sample in the diagnosis of tuberculosis. In certain groups of individuals who cannot produce sputum, for example, children, non-sputum-based methods have particular importance. We found that the method tested was able to detect bacterial killing by active antibiotics that disrupt the cell wall and lead to fragmentation of bacteria. However, the assay can't detect inactive bacteria or bacteria that are active with an intact cell wall.

The treatment of drug-resistant Mycobacterium tuberculosis relies on complex antibiotic therapy. Inadequate antibiotic exposure can lead to treatment failure, acquired drug resistance, and an increased risk of adverse events. Therapeutic drug monitoring (TDM) can be used to optimize the antibiotic exposure. Therefore, we aimed to develop a single-run multiplex assay using high-performance liquid chromatography–mass spectrometry (HPLC–MS) for TDM of patients with multidrug-resistant, pre-extensively drug-resistant and extensively drug-resistant tuberculosis. A target profile for sufficient performance, based on the intended clinical application, was established and the assay was developed accordingly. Antibiotics were analyzed on a zwitterionic hydrophilic interaction liquid chromatography column and a triple quadrupole mass spectrometer using stable isotope-labeled internal standards. The assay was sufficiently sensitive to monitor drug concentrations over five half-lives for rifampicin, rifabutin, levofloxacin, moxifloxacin, bedaquiline, linezolid, clofazimine, terizidone/cycloserine, ethambutol, delamanid, pyrazinamide, meropenem, prothionamide, and para-amino salicylic acid (PAS). Accuracy and precision were sufficient to support clinical decision making (≤±15% in clinical samples and ±20–25% in spiked samples, with 80% of future measured concentrations predicted to fall within ±40% of nominal concentrations). The method was applied in the TDM of two patients with complex drug-resistant tuberculosis. All relevant antibiotics from their regimens could be quantified and high-dose therapy was initiated, followed by microbiological conversion. In conclusion, we developed a multiplex assay that enables TDM of the relevant first- and second-line anti-tuberculosis medicines in a single run and was able to show its applicability in TDM of two drug-resistant tuberculosis patients.

Tuberculosis (TB) is an infectious disease that affects millions of people globally, with 1.6 million deaths annually. TB treatment requires combinations of multiple different antibiotics for many months, and toxic side effects can occur.

ABSTRACT Mycobacterium tuberculosis is well adapted to survive and persist in the infected host, escaping the host immune response. Since polyamines, which are synthesized by infected macrophages are able to inhibit the growth of M. tuberculosis , the pathogen needs strategies to cope with toxic spermine. The actinomycete Streptomyces coelicolor , closely related to M. tuberculosis makes use of a gamma-glutamylation pathway to functionally neutralize spermine. We therefore considered whether a similar pathway would be functional in M. tuberculosis . In the current study we demonstrated that M. tuberculosis growth was inhibited by the polyamine spermine. Using a glutamine synthetase-based in vitro enzymatic activity assay we determined that GlnA3 Mt (Rv1878) is a gamma-glutamylspermine synthetase. In an in vitro phosphate release assay we showed that purified His-Strep-GlnA3 Mt as well as native GlnA3 Mt prefer spermine as a substrate to putrescine, cadaverine, spermidine or other monoamines and amino acids, suggesting that GlnA3 Mt may play a specific role in the detoxification of the polyamine spermine. However, the deletion of the glnA3 gene in M. tuberculosis did not result in growth inhibition or enhanced sensitivity of M. tuberculosis in the presence of high spermine concentrations. Subsequent RNAsequencing of M. tuberculosis bacteria revealed that the gene cluster consisting of the efflux pump-encoding rv3065-rv3066-rv3067 genes is upregulated upon spermine treatment, suggesting its involvement in bacterial survival under elevated spermine concentrations. IMPORTANCE Antibiotics for the treatment of Mycobacterium tuberculosis infections attack classical bacterial targets, such as the cell envelope or the ribosome. Upon M. tuberculosis infection macrophages synthesize the polyamine spermine which - at elevated concentrations - is toxic for M. tuberculosis . Based on our investigations of spermine resistance in the closely related actinomycete Streptomyces coelicolor , we hypothesized that the glutamyl-sperminesynthetase GlnA3 may be responsible for resistance against toxic spermine. Here we show that the mycobacterial glutamyl-sperminesynthetase indeed can inactivate spermine by glutamylation. However, GlnA3 is probably not the only resistance mechanism since a glnA3 mutant of M. tuberculosis can survive under spermine stress. Gene expression studies suggest that an efflux pump may participate in resistance. The functional role of GlnA3 Mt as well as of the spermine transporter in the pathogenicity of M. tuberculosis is of special interest for their validation as new targets of novel anti-tubercular drugs.

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(tg)) mice develop a TB pathology very similar to patients and, in contrast to other mouse models, also share pathogenetic mechanisms. Accordingly, IL-13(tg) animals represent an ideal model for analyzing the penetration of novel anti-TB drugs into various compartments of necrotic granulomas by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MS imaging). In the present study, we evaluated the suitability of BALB/c IL-13(tg) mice for determining the antibiotic distribution within necrotizing lesions. To this end, we established a workflow based on the inactivation of M. tuberculosis by gamma irradiation while preserving lung tissue integrity and drug distribution, which is essential for correlating drug penetration with lesion pathology. MALDI-MS imaging analysis of clofazimine, pyrazinamide, and rifampicin revealed a drug-specific distribution within different lesion types, including cellular granulomas, developing in BALB/c wild-type mice, and necrotic granulomas in BALB/c IL-13(tg) animals, emphasizing the necessity of preclinical models reflecting human pathology. Most importantly, our study demonstrates that BALB/c IL-13(tg) mice recapitulate the penetration of antibiotics into human lesions. Therefore, our workflow in combination with the IL-13(tg) mouse model provides an improved and accelerated evaluation of novel anti-TB drugs and new regimens in the preclinical stage.

Inappropriately high breakpoints have resulted in systematic false-susceptible AST results to anti-TB drugs. MIC, PK/PD and clinical outcome data should be combined when setting breakpoints to minimise the emergence and spread of antimicrobial resistance.https://bit.ly/3i43wb6

BACKGROUND:Pulmonary Tuberculosis (TB) is diagnosed through sputum samples. As sputum sampling is challenging in children and cachexic patients, the development of diagnostic tests using saliva appears promising but has been discouraged due to low bacterial load and poor sensitivity. Here, we present a novel and rapid method to enrich Mycobacterium tuberculosis (Mtb) from saliva, which may serve as a basis for a diagnostic saliva test.METHODS:Lipobiotin-functionalized magnetic beads (LMBs) were incubated with Mtb-spiked PBS and saliva from healthy donors as well as with saliva from TB patients. Flow cytometry was used to evaluate the capacity of the beads to bind Mtb, while real-time quantitative polymerase chain reaction (qPCR) was utilized to detect Mtb and determine the amount of mycobacterial DNA in different sample types.RESULTS:We found that LMBs bind Mtb efficiently when compared to non-functionalized beads. The development of an qPCR assay based on the use of LMBs (LMB assay) allowed us to enrich mycobacterial DNA in spiked sample types, including PBS and saliva from healthy donors (enrichment of up to ~8.7 fold). In Mtb-spiked saliva samples, we found that the LMB assay improved the detection rate of 102 bacteria in a volume of 5 ml from 0 out of 15 (0%) to 6 out of 15 (40%). Consistent with that, the LMB assay increased the rate of correctly identified saliva samples from TB patients in two independent cohorts.CONCLUSIONS:Implementation of the principle of the LMB-based assay may improve the sensitivity of existing diagnostic techniques, e.g. by functionalizing materials that facilitate Mtb sampling from the oral cavity.

Close to one quarter of the world’s population is infected with Mycobacterium tuberculosis (Mtb). Tuberculosis (TB) is hard to treat and causes 3,800 deaths each day. Multidrug-resistant TB (MDR-TB) is a global public health threat. Rapid identification and drug susceptibility testing (DST) of Mtb is key to prevent the spread of MDR-TB. We studied whether single-cell Raman spectroscopy is suitable to provide reliable species identification (ID) and DST results while significantly accelerating the time-to-result (TTR) compared to the standard of care (SoC), that is PCR-based species ID within one day and phenotypic DST results at 7-10 days of culture-positivity.

BackgroundIn 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.ObjectiveTo 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.MethodsHospitalization 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.ResultsHospitalization 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.ConclusionHigher 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.

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.

We surveyed availability of phenotypic drug susceptibility testing for drug-resistant Mycobacterium tuberculosis in Europe. Of 27 laboratories, 17 tested for linezolid, 11 for clofazimine, 9 for bedaquiline, and 6 for delamanid during 2019. Our findings indicate that testing capacity for newer and repurposed tuberculosis drugs exists, but its availability is limited.

Therapeutic drug monitoring (TDM) has been suggested to improve treatment responses in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB), but evidence is still scarce. We share our experience with TDM in anti-tuberculosis therapy in a patient with very advanced XDR-TB. A 34-year-old male from Ukraine was admitted to our hospital in November 2018 for the treatment of tuberculosis. He had been diagnosed in December 2008 and had since received 8 treatment episodes in Ukraine with relapses and failing therapies. On admission, time to positivity for Mycobacterium tuberculosis in liquid sputum culture was 7 days. Phenotypic drug susceptibility testing revealed drug-resistance to all 1st- and 2nd-line anti-tuberculosis drugs apart from terizidone, delamanid, and pretomanid. Under treatment with 800 mg moxifloxacin and standard doses of bedaquiline, terizidone, delamanid, and meropenem/amoxiclav, no sustainable culture conversion could be achieved over five months. In contrast, a TDM-controlled high-dose treatment, including i.a. 1600 mg moxifloxacin, bedaquiline 300 mg thrice/week, and 1000 mg terizidone, induced culture negativity that was stable over 15 months. However, a subsequent attempt to stop therapy resulted in a microbiological relapse and necessitated a therapy re-start, now under even further intensified treatment including i.a. bedaquiline 400 mg thrice/week, 9.6 g meropenem, and 7.2 g amoxiclav per day. This regimen quickly led to culture conversion again. There were no serious adverse events or acquired drug resistances observed. In summary, TDM-guided therapy can help to overcome low-intermediate level resistance in M/XDR-TB. However, extended treatment duration might be needed to achieve cure.

Atypical mycobacterial panniculitis was diagnosed in a cat. Mycobacterium setense was identified as causative agent by 16 S rRNA gene sequence analysis. This a gram-positive rod-shaped acid-fast bacterium belonging to Mycobacterium fortuitum group was never reported before in diseased animals. Resistance to doxycycline and clarithromycin was detected. During treatment with pradofloxacin, additional resistance to fluoroquinolones developed which was due to a mutation in the gyrase gene gyrA (S90W exchange). Despite of antimicrobial treatment for 33 months the patient did not fully recover. Species identification and susceptibility testing for choosing adequate antimicrobial treatment is recommended in cases of feline mycobacterial panniculitis.

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.

Sustainable support from healthcare bodies is needed to preserve TB laboratory capacity, and maintain personnel and skills, to minimise negative effects of the COVID-19 pandemic on laboratory services severely disrupted in the early months of pandemichttps://bit.ly/38camaL