Susceptibility testing breakpoints for Mycobacterium tuberculosis categorize isolates with resistance mutations in gyrA as susceptible to fluoroquinolones: implications for MDR-TB treatment and the definition of XDR-TB.

OBJECTIVES: Fluoroquinolones (FQs) are important in the treatment of MDR-TB and in the definition of XDR-TB. Our objective was to investigate how discrepancies in the phenotypic and genotypic methods for antimicrobial susceptibility testing could affect the interpretation of antimicrobial susceptibility test results. METHODS: We analysed MICs of ofloxacin and levofloxacin in Middlebrook 7H10 broth (7H10) as well as sequencing of the quinolone resistance-determining region of the gyrA gene and the MTBDRsl assay in 75 resistant isolates, including MDR and XDR strains of Mycobacterium tuberculosis. RESULTS: Among 75 resistant isolates, 27 had mutations associated with FQ resistance. Among isolates with resistance mutations in gyrA, 26% (seven of 27) were susceptible to levofloxacin and ofloxacin by phenotypic testing at 1 mg/L and 2 mg/L. The most common mutation was in codon 94 and these isolates had significantly increased MICs of levofloxacin (2-8 mg/L) compared with isolates with mutations in codon 90 (0.25-2 mg/L, P < 0.05). The sensitivity and specificity for the MTBDRsl assay compared with gyrA sequencing were 96% and 98%, respectively. CONCLUSION: Current critical concentrations may classify up to 26% of isolates with gyrA mutations as susceptible to FQs due to a close relationship between susceptible and resistant populations. These results should be considered while improving clinical breakpoints for M. tuberculosis and may have an impact on the definition of XDR-TB.

The geographic diversity of nontuberculous mycobacteria isolated from pulmonary samples: an NTM-NET collaborative study.

A significant knowledge gap exists concerning the geographical distribution of nontuberculous mycobacteria (NTM) isolation worldwide. To provide a snapshot of NTM species distribution, global partners in the NTM-Network European Trials Group (NET) framework (www.ntm-net.org), a branch of the Tuberculosis Network European Trials Group (TB-NET), provided identification results of the total number of patients in 2008 in whom NTM were isolated from pulmonary samples. From these data, we visualised the relative distribution of the different NTM found per continent and per country. We received species identification data for 20 182 patients, from 62 laboratories in 30 countries across six continents. 91 different NTM species were isolated. Mycobacterium avium complex (MAC) bacteria predominated in most countries, followed by M. gordonae and M. xenopi. Important differences in geographical distribution of MAC species as well as M. xenopi, M. kansasii and rapid-growing mycobacteria were observed. This snapshot demonstrates that the species distribution among NTM isolates from pulmonary specimens in the year 2008 differed by continent and differed by country within these continents. These differences in species distribution may partly determine the frequency and manifestations of pulmonary NTM disease in each geographical location.

Challenging a dogma: antimicrobial susceptibility testing breakpoints for Mycobacterium tuberculosis.

The rise in multidrug-resistant tuberculosis makes it increasingly important that antimicrobial susceptibility testing of Mycobacterium tuberculosis produce clinically meaningful and technically reproducible results. Unfortunately, this is not always the case because mycobacteriology specialists have not followed generally accepted modern principles for the establishment of susceptibility breakpoints for bacterial and fungal pathogens. These principles specifically call for a definition of the minimum inhibitory concentrations (MICs) applicable to organisms without resistance mechanisms (also known as wild-type MIC distributions), to be used in combination with data on clinical outcomes, pharmacokinetics and pharmacodynamics. In a series of papers the authors have defined tentative wild-type MIC distributions for M. tuberculosis and hope that other researchers will follow their example and provide confirmatory data. They suggest that some breakpoints are in need of revision because they either (i) bisect the wild-type distribution, which leads to poor reproducibility in antimicrobial susceptibility testing, or (ii) are substantially higher than the MICs of wild-type organisms without supporting clinical evidence, which may result in some strains being falsely reported as susceptible. The authors recommend, in short, that susceptibility breakpoints for antituberculosis agents be systematically reviewed and revised, if necessary, using the same modern tools now accepted for all other bacteria and fungi by the scientific community and by the European Medicines Agency and the European Centre for Disease Prevention and Control. For several agents this would greatly improve the accuracy and reproducibility of antimicrobial susceptibility testing of M. tuberculosis.

[Jaundice in the adult patient]. / ABC om Ikterus hos vuxna.

Jaundice is an alarm symptom that should always be treated urgently, regardless of whether the responsible doctor is in primary care or in the emergency room. The differential diagnoses can be significantly delimited (hepatocellular vs. cholestasis) and several clues to the etiology can be determined from a carefully performed anamnesis, clinical examination, and basic laboratory tests. Exclusion of cholestatic etiology is essential due to life-threatening differential diagnoses and complications, but acute medical conditions also occur, such as acute liver failure. Prompt processing at the correct instance can be crucial for the short-term and long-term prognosis of the patient.

Stockholm score of lesion detection on computed tomography following mild traumatic brain injury (SELECT-TBI): study protocol for a multicentre, retrospective, observational cohort study.

INTRODUCTION: Mild traumatic brain injury (mTBI) is one of the most common reasons for emergency department (ED) visits. A portion of patients with mTBI will develop an intracranial lesion that might require medical or surgical intervention. In these patients, swift diagnosis and management is paramount. Several guidelines have been developed to help direct patients with mTBI for head CT scanning, but they lack specificity, do not consider the interactions between risk factors and do not provide an individualised estimate of intracranial lesion risk. The aim of this study is to create a model that estimates individualised intracranial lesion risks in patients with mTBI who present to the ED. METHODS AND ANALYSIS: This will be a retrospective cohort study conducted at ED hospitals in Stockholm, Sweden. Eligible patients are adults (≥15 years) with mTBI who presented to the ED within 24 hours of injury and performed a CT scan. The primary outcome will be a traumatic lesion on head CT. The secondary outcomes will be any clinically significant lesion, defined as an intracranial finding that led to neurosurgical intervention, hospital admission ≥48 hours due to TBI or death due to TBI. Machine-learning models will be applied to create scores predicting the primary and secondary outcomes. An estimated 20 000 patients will be included. ETHICS AND DISSEMINATION: The study has been approved by the Swedish Ethical Review Authority (Dnr: 2020-05728). The research findings will be disseminated through peer-reviewed scientific publications and presentations at international conferences. TRIAL REGISTRATION NUMBER: NCT04995068.

CD4+ T cell proliferative responses to PPD and CFP-10 associate with recent M. tuberculosis infection.

Interferon-γ release assays cannot differentiate latent from active tuberculosis (TB), nor identify the recently infected with increased risk of active disease. The objective of this study was to identify biomarkers of recent infection following exposure to tuberculosis, to increase the positive predictive value for incipient TB. Contacts to patients with pulmonary TB were tested repeatedly with interferon-γ release assays and flow-cytometry. Proliferative CD4+ T cell responses to purified protein derivative (PPD) and 11 M. tuberculosis antigens were analysed. The individual probability of recent and remote infection was estimated using clinical data in a novel mathematical model and compared with CD4+ responses in a prediction model. The most specific prediction of recent infection was high CD4+ proliferative responses to CFP-10 and PPD and a low CD4+ response to ESAT-6. CD4+ proliferative responses to Rec85a, Rec85b and Rv1284 were also observed in recent infection, but did not reach significance in the prediction model. CONCLUSIONS: High CD4+ proliferative responses to CFP-10 and PPD and a low response to ESAT-6 may be used as biomarkers to improve positive predictive values for recent LTBI and thus, increased risk of incipient TB. Rec85a, Rec85b and Rv1284 are also of interest to study further in this context.

Phylogenetically informative mutations in genes implicated in antibiotic resistance in Mycobacterium tuberculosis complex.

BACKGROUND: A comprehensive understanding of the pre-existing genetic variation in genes associated with antibiotic resistance in the Mycobacterium tuberculosis complex (MTBC) is needed to accurately interpret whole-genome sequencing data for genotypic drug susceptibility testing (DST). METHODS: We investigated mutations in 92 genes implicated in resistance to 21 anti-tuberculosis drugs using the genomes of 405 phylogenetically diverse MTBC strains. The role of phylogenetically informative mutations was assessed by routine phenotypic DST data for the first-line drugs isoniazid, rifampicin, ethambutol, and pyrazinamide from a separate collection of over 7000 clinical strains. Selected mutations/strains were further investigated by minimum inhibitory concentration (MIC) testing. RESULTS: Out of 547 phylogenetically informative mutations identified, 138 were classified as not correlating with resistance to first-line drugs. MIC testing did not reveal a discernible impact of a Rv1979c deletion shared by M. africanum lineage 5 strains on resistance to clofazimine. Finally, we found molecular evidence that some MTBC subgroups may be hyper-susceptible to bedaquiline and clofazimine by different loss-of-function mutations affecting a drug efflux pump subunit (MmpL5). CONCLUSIONS: Our findings underline that the genetic diversity in MTBC has to be studied more systematically to inform the design of clinical trials and to define sound epidemiologic cut-off values (ECOFFs) for new and repurposed anti-tuberculosis drugs. In that regard, our comprehensive variant catalogue provides a solid basis for the interpretation of mutations in genotypic as well as in phenotypic DST assays.

Evaluation of wild-type MIC distributions as a tool for determination of clinical breakpoints for Mycobacterium tuberculosis.

OBJECTIVES: The aim of this study was to establish wild-type MIC distributions of first-line drugs for Mycobacterium tuberculosis, as well as to explore the usefulness of such distributions when setting clinical breakpoints. METHODS: We determined the MICs of rifampicin, isoniazid and ethambutol for M. tuberculosis using a Middlebrook 7H10 dilution method for 90 consecutive clinical isolates, 8 resistant strains and 16 isolates from the WHO proficiency test panel. M. tuberculosis H37Rv was used for quality control and susceptibility results using 7H10 were compared with the results obtained with BACTEC460. RESULTS: The agreement with BACTEC460 was very high for isoniazid (99.1%) and rifampicin (99.1%) but lower for ethambutol (94.7%). Intra- and inter-assay variation was below one MIC dilution. The MIC distributions for isoniazid and rifampicin provided a clear separation between susceptible and resistant strains. Regarding ethambutol, the current breakpoint for 7H10 (5 mg/L) is close to the wild-type and all strains (n = 6) showing a disagreement between BACTEC460 and 7H10 were distributed very close to the breakpoint (MIC 4-8 mg/L). This problematic relation was confirmed by investigating isolates from the WHO panel with an agreement <95% (64%-88% among 26 laboratories, n = 4) for which the MICs were 4-8 mg/L. CONCLUSIONS: Utilizing the wild-type MIC distribution was found to be as useful in M. tuberculosis as in other bacteria when setting clinical breakpoints. We suggest that the present clinical breakpoints should be re-evaluated, taking into account wild-type MIC distributions and available pharmacokinetic data.

The nitrate reductase assay for the rapid detection of isoniazid and rifampicin resistance in Mycobacterium tuberculosis: a systematic review and meta-analysis.

OBJECTIVES: The reference standard methods for drug susceptibility testing (DST) of M. tuberculosis are very slow to give results, and due to the emergence of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis, there is an urgent demand for new, rapid and accurate DST methods, particularly in low-income countries. The nitrate reductase assay (NRA) has been proposed as a rapid method for the detection of resistance to rifampicin and isoniazid, but its accuracy has not been systematically evaluated. METHODS: We performed a systematic review and meta-analysis to evaluate the accuracy of the NRA for the detection of rifampicin- and isoniazid-resistant tuberculosis. We searched Medline PubMed (NCBI), Global Health-CAB, EJS-E (EbscoHost), ISI Web, Web of Science and IFCC and contacted authors if additional information was required. Fifteen studies met our inclusion criteria for rifampicin resistance detection and 13 for isoniazid. Of these, the majority of the studies used culture isolates on solid medium, four used culture isolates on liquid medium and three used sputum samples. We applied the summary receiver operating characteristic (SROC) curve to perform meta-analysis and to summarize diagnostic accuracy. RESULTS: For rifampicin, the majority of the studies that applied NRA to isolates had a sensitivity and specificity >94% and for isoniazid, >92%. The three studies that applied NRA directly on sputum samples had a sensitivity and specificity that ranged between 88% and 100%. The SROC curve had an area of >0.99 for both drugs. CONCLUSIONS: There is evidence that NRA is highly sensitive and specific for the rapid detection of rifampicin and isoniazid resistance in culture isolates. More evidence is required for the NRA applied directly on sputum samples, but preliminary results appear promising and show a good sensitivity and specificity. Additional studies are required in countries with a high prevalence of MDR-TB and also cost-effectiveness analysis in order to obtain a complete picture on the utility of this method for rapid drug resistance detection in tuberculosis.

A luciferase-based assay for rapid assessment of drug activity against Mycobacterium tuberculosis including monitoring of macrophage viability.

The intracellular (IC) effect of drugs against Mycobacterium tuberculosis (Mtb) is not well established but increasingly important to consider when combining current and future multidrug regimens into the best possible treatment strategies. For this purpose, we developed an IC model based on a genetically modified Mtb H37Rv strain, expressing the Vibrio harvei luciferase (H37Rv-lux) infecting the human macrophage like cell line THP-1. Cells were infected at a low multiplicity of infection (1:1) and subsequently exposed to isoniazid (INH), ethambutol (EMB), amikacin (AMI) or levofloxacin (LEV) for 5days in a 96-well format. Cell viability was evaluated by Calcein AM and was maintained throughout the experiment. The number of viable H37Rv-lux was determined by luminescence and verified by a colony forming unit analysis. The results were compared to the effects of the same drugs in broth cultures. AMI, EMB and LEV were significantly less effective intracellularly (MIC90: >4mg/L, 8mg/L and 2mg/L, respectively) compared to extracellularly (MIC90: 0.5mg/L for AMI and EMB; 0.25mg/L for LEV). The reverse was the case for INH (IC: 0.064mg/L vs EC: 0.25mg/L). In conclusion, this luciferase based method, in which monitoring of cell viability is included, has the potential to become a useful tool while evaluating the intracellular effects of anti-mycobacterial drugs.

A 24-well plate assay for simultaneous testing of first and second line drugs against Mycobacterium tuberculosis in a high endemic setting.

BACKGROUND: Early detection of drug resistance is one of the priorities of tuberculosis (TB) control programs as drug resistance is increasing. New molecular assays are only accessible for a minority of the second line drugs and their availability in high endemic settings is also hampered by high cost and logistic challenges. Therefore, we evaluated a previously developed method for drug susceptibility testing (DST) including both first- and second line anti-TB drugs for use in high endemic areas. RESULTS: Baseline mycobacterial isolates from 78 consecutive pulmonary TB patients from Addis Ababa, Ethiopia who were culture positive for Mycobacterium tuberculosis at the end of a two-month directly observed treatment short course (DOTS) were included. The isolates were simultaneously tested for isoniazid, rifampicin, ethambutol, streptomycin, amikacin, kanamycin, capreomycin, ofloxacin, moxifloxacin, ethionamide and para-aminosalicylic acid susceptibility using the indirect proportion method adopted for 24-well agar plates containing Middlebrook 7H10 medium. Applying the 24-well plate assay, 43 (55.1%) isolates were resistant to one or more of the first line drugs tested (isoniazid, rifampicin and ethambutol). MDR-TB was identified in 20.5% of this selected group and there was a perfect correlation for rifampicin resistance with the results from the genotype MTBDRplus assay. All isolates were susceptible to aminoglycosides and fluoroquinolones in agreement with the genotype MTBDRsl assay. The only tested second line drug associated to resistance was ethionamide (14.1% resistant). The method was reproducible with stable results for internal controls (one multi-drug resistant (MDR) and one pan-susceptible strain (H37Rv) and DST results could be reported at two weeks. CONCLUSIONS: The 24-well plate method for simultaneous DST for first- and second line drugs was found to be reproducible and correlated well to molecular drug susceptibility tests. It is likely to be useful in high-endemic areas for surveillance as well as for the detection of second line drug resistance in targeted groups such as in those who fail empirical MDR treatment.

The GenoType® MTBDRplus assay for detection of drug resistance in Mycobacterium tuberculosis in Sweden.

The performance of the GenoType(®) MTBDRplus assay was compared with conventional drug susceptibility testing (DST) in 604 patients with tuberculosis. The study comprised 477 Mycobacterium tuberculosis complex isolates and 127 preparations of DNA from clinical specimens which had been tested positive for M. tuberculosis by COBAS(®)TaqMan(®) 48. By DST, isoniazid (INH) monoresistance was diagnosed in 56 (9.3%), rifampicin (RMP) monoresistance in 2 (0.3%) and multidrug resistance (MDR) in 21 (3.5%) of the cases. The sensitivity of the MTBDRplus assay was 87.5%, 100% and 95.2% for INH resistance, RMP resistance and MDR respectively. The specificity was 100% for all resistance patterns. The dominating mutations in RMP and INH resistant isolates were in codon 531 of the rpoB gene and codon 315 of the KatG gene. The turnaround time for detection of drug resistance can be shortened from a median of 21 days for DST to 7 days for the MTBDRplus assay. This may have a significant impact on routine work flow of a mycobacteriology laboratory.

Tentative susceptibility testing breakpoint for the neuroleptic drug thioridazine, a treatment option for multi- and extensively drug resistant tuberculosis.

INTRODUCTION: New drugs against multi-(MDR) and extensively drug (XDR) resistant tuberculosis are urgently needed. While new candidate drugs are being developed, reinvestigation of already approved drugs available for other indications could be of value. The objective of this study is to determine tentative drug susceptibility testing strategies and breakpoints for thioridazine, a well-known and well-tolerated neuroleptic drug, which has been shown to be effective against drug resistant tuberculosis both in vitro and in vivo. METHODS: By testing the minimal inhibitory concentration (MIC) on Middlebrook 7H10 media, the wild-type distribution of thioridazine was established for Mycobacterium tuberculosis (n=51) and this distribution was compared to the MICs of M/XDR strains (n=67). RESULTS: A tentative epidemiological cut off (ECOFF) of thioridazine at 16mg/L was suggested. Even though such concentrations are not clinically achievable in serum, thioridazine is concentrated intracellularly and concentrations of only 0.1mg/L has been shown to kill M. tuberculosis residing inside cells. MICs above the wild-type (MIC>16mg/L) were found in 4/67 (6%) of the M/XDR strains suggesting that resistance mechanisms against thioridazine may already be present in resistant clinical strains. CONCLUSIONS: In view of the difficulties obtaining clinical outcome data for single drugs in the case of tuberculosis since combination therapy is mandatory, the tentative ECOFF may be considered a tentative clinical breakpoint, but the findings should be validated by others. The data from this study strengthens the use of thioridazine as a treatment option for M/XDR tuberculosis, although its proper place in the therapeutic arsenal should ideally be confirmed in clinical trials.

Resistance to first-line anti-TB drugs is associated with reduced nitric oxide susceptibility in Mycobacterium tuberculosis.

BACKGROUND AND OBJECTIVE: The relative contribution of nitric oxide (NO) to the killing of Mycobacterium tuberculosis in human tuberculosis (TB) is controversial, although this has been firmly established in rodents. Studies have demonstrated that clinical strains of M. tuberculosis differ in susceptibility to NO, but how this correlates to drug susceptibility and clinical outcome is not known. METHODS: In this study, 50 sputum smear- and culture-positive patients with pulmonary TB in Gondar, Ethiopia were included. Clinical parameters were recorded and drug susceptibility profile and spoligotyping patterns were investigated. NO susceptibility was studied by exposing the strains to the NO donor DETA/NO. RESULTS: Clinical isolates of M. tuberculosis showed a dose- and time-dependent response when exposed to NO. The most frequent spoligotypes found were CAS1-Delhi and T3_ETH in a total of nine known spoligotypes and four orphan patterns. There was a significant association between reduced susceptibility to NO (>10% survival after exposure to 1 mM DETA/NO) and resistance against first-line anti-TB drugs, in particular isoniazid (INH). Patients infected with strains of M. tuberculosis with reduced susceptibility to NO showed no difference in cure rate or other clinical parameters but a tendency towards lower rate of weight gain after two months of treatment, independent of antibiotic resistance. CONCLUSION: There is a correlation between resistance to first-line anti-TB drugs and reduced NO susceptibility in clinical strains of M. tuberculosis. Further studies including the mechanisms of reduced NO susceptibility are warranted and could identify targets for new therapeutic interventions.

Antioxidants protect keratinocytes against M. ulcerans mycolactone cytotoxicity.

BACKGROUND: Mycobacterium ulcerans is the causative agent of necrotizing skin ulcerations in distinctive geographical areas. M. ulcerans produces a macrolide toxin, mycolactone, which has been identified as an important virulence factor in ulcer formation. Mycolactone is cytotoxic to fibroblasts and adipocytes in vitro and has modulating activity on immune cell functions. The effect of mycolactone on keratinocytes has not been reported previously and the mechanism of mycolactone toxicity is presently unknown. Many other macrolide substances have cytotoxic and immunosuppressive activities and mediate some of their effects via production of reactive oxygen species (ROS). We have studied the effect of mycolactone in vitro on human keratinocytes--key cells in wound healing--and tested the hypothesis that the cytotoxic effect of mycolactone is mediated by ROS. METHODOLOGY/PRINCIPAL FINDINGS: The effect of mycolactone on primary skin keratinocyte growth and cell numbers was investigated in serum free growth medium in the presence of different antioxidants. A concentration and time dependent reduction in keratinocyte cell numbers was observed after exposure to mycolactone. Several different antioxidants inhibited this effect partly. The ROS inhibiting substance deferoxamine, which acts via chelation of Fe(2+), completely prevented mycolactone mediated cytotoxicity. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that mycolactone mediated cytotoxicity can be inhibited by deferoxamine, suggesting a role of iron and ROS in mycolactone induced cytotoxicity of keratinocytes. The data provide a basis for the understanding of Buruli ulcer pathology and the development of improved therapies for this disease.