Operationalising targeted next-generation sequencing for routine diagnosis of drug-resistant TB.

BACKGROUND: Phenotypic drug susceptibility testing (pDST) for Mycobacterium tuberculosis can take up to 8 weeks, while conventional molecular tests identify a limited set of resistance mutations. Targeted next-generation sequencing (tNGS) offers rapid results for predicting comprehensive drug resistance, and this study sought to explore its operational feasibility within a public health laboratory in Mumbai, India. METHODS: Pulmonary samples from consenting patients testing Xpert MTB-positive were tested for drug resistance by conventional methods and using tNGS. Laboratory operational and logistical implementation experiences from study team members are shared below. RESULTS: Of the total number of patients tested, 70% (113/161) had no history of previous TB or treatment; however, 88.2% (n = 142) had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB). There was a high concordance between resistance predictions of tNGS and pDST for most drugs, with tNGS more accurately identifying resistance overall. tNGS was integrated and adapted into the laboratory workflow; however, batching samples caused significantly longer result turnaround time, fastest at 24 days. Manual DNA extraction caused inefficiencies; thus protocol optimisations were performed. Technical expertise was required for analysis of uncharacterised mutations and interpretation of report templates. tNGS cost per sample was US$230, while for pDST this was US$119. CONCLUSIONS: Implementation of tNGS is feasible in reference laboratories. It can rapidly identify drug resistance and should be considered as a potential alternative to pDST.

CONTEXTE: Les tests phénotypiques de sensibilité aux médicaments (pDST) pour Mycobacterium tuberculosis peuvent prendre jusqu'à 8 semaines, tandis que les tests moléculaires conventionnels identifient un ensemble limité de mutations de résistance. Le séquençage ciblé de la prochaine génération (tNGS) offre des résultats rapides pour prédire la résistance globale aux médicaments, et cette étude avait pour objectif d'explorer sa faisabilité opérationnelle au sein d'un laboratoire de santé publique à Mumbai, en Inde. MÉTHODES: Des échantillons pulmonaires de patients consentants testés positifs au Xpert MTB ont été testés pour la résistance aux médicaments par des méthodes conventionnelles et en utilisant le tNGS. Les expériences des membres de l'équipe de l'étude en matière de fonctionnement du laboratoire et de mise en œuvre logistique sont présentées ci-dessous. RÉSULTATS: Sur le nombre total de patients testés, 70% (113/161) n'avaient pas d'antécédents de TB ou de traitement ; cependant, 88,2% (n = 142) présentaient une TB résistante à la rifampicine/multirésistante aux médicaments (RR/MDR-TB). La concordance entre les prédictions de résistance de la tNGS et de la pDST était élevée pour la plupart des médicaments, la tNGS identifiant globalement la résistance avec plus de précision. La tNGS a été intégrée et adaptée au flux de travail du laboratoire ; toutefois, la mise en lots des échantillons a entraîné un délai d'obtention des résultats beaucoup plus long, le plus rapide étant de 24 jours. L'extraction manuelle de l'ADN a été source d'inefficacité ; le protocole a donc été optimisé. L'analyse des mutations non caractérisées et l'interprétation des modèles de rapport ont nécessité une expertise technique. Le coût du tNGS par échantillon s'élevait à US$230, contre US$119 pour le pDST. CONCLUSIONS: La mise en œuvre de la tNGS est possible dans les laboratoires de référence. Elle permet d'identifier rapidement la résistance aux médicaments et devrait être considérée comme une alternative potentielle à la pDST.

Clinical utility of target-based next-generation sequencing for drug-resistant TB.

BACKGROUND: In high TB burden countries, access to drug susceptibility testing is a major bottleneck. Targeted next-generation sequencing (tNGS) is a promising technology for rapid resistance detection. This study assessed the role of tNGS for the diagnosis of drug-resistant TB (DR-TB).METHODS: A total of 161 samples from bacteriologically confirmed TB cases were subjected to tNGS using the Deeplex® Myc-TB kit and sequenced using the MiSeq platform. These samples were also processed for conventional phenotypic DST (pDST) using 13 drugs on Mycobacteria Growth Indicator Tube and line-probe assays (MTBDRplus and MTBDRsl).RESULTS: There were 146 DR-TB and 15 drug-susceptible TB (DS-TB) samples. About 70% of patients with DR-TB had no previous TB treatment history. Overall, 88.2% had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB), 58.5% pre-extensively drug-resistant TB (pre-XDR-TB) and 9.2% had XDR-TB as defined by the WHO (2020). Around 8% (n = 13) of samples were non-culturable; however, identified 8 were resistant to first and second-line drugs using tNGS. Resistance frequency was similar across methods, with discordance in drugs less reliable using pDST or with limited mutational representation within databases. Sensitivities were aligned with literature reports for most drugs. We observed 10% heteroresistance, while 75% of strains were of Lineages 2 and 3.CONCLUSIONS: Programme data supported tNGS in the diagnosis of DR-TB for early treatment using individualised regimens.

Identification of novel inhibitors targeting TIRAP interactions with BTK and PKCδ in inflammation through an in silico approach.

Advanced computational tools focusing on protein-protein interaction (PPI) based drug development is a powerful platform to accelerate the therapeutic development of small lead molecules and repurposed drugs. Toll/interleukin-1 receptor (TIR) domain-containing adapter protein (TIRAP) and its interactions with other proteins in macrophages signalling are crucial components of severe or persistent inflammation. TIRAP activation through Bruton's tyrosine kinase (BTK) and Protein Kinase C delta (PKCδ) is essential for downstream inflammatory signalling. We created homology-based structural models of BTK and PKCδ in MODELLER 9.24. TIRAP interactions with BTK and PKCδ in its non-phosphorylated and phosphorylated states were determined by multiple docking tools including HADDOCK 2.4, pyDockWEB and ClusPro 2.0. Food and Drug Administration (FDA)-approved drugs were virtually screened through Discovery Studio LibDock and Autodock Vina tools to target the common TIR domain residues of TIRAP, which interact with both BTK and PKC at the identified interfacial sites of the complexes. Four FDA-approved drugs were identified and found to have stable interactions over a range of 100 ns MD simulation timescales. These drugs block the interactions of both kinases with TIRAP in silico. Hence, these drugs have the potential to dampen downstream inflammatory signalling and inflammation-mediated disease.

Feasibility of radiation dose reduction using AIDR-3D in dynamic pulmonary CT perfusion.

AIM: To assess the feasibility of radiation dose reduction with adaptive iterative dose reduction (AIDR-6 3D) reconstruction in dynamic pulmonary CT perfusion. MATERIALS AND METHODS: CTP examinations of 10 patients acquired at 100 kVp/50 mAs were reconstructed with filtered back projection (FBP) and AIDR-3D. Artificial noise was added to raw data (pre-reconstruction projection data) to simulate lower tube current scanning. Radiodensity (in Hounsfield units), noise, and perfusion values were compared. RESULTS: There was no significant difference in noise between the full and simulated reduced tube current with AIDR-3D reconstruction (p = 1). There was significantly lower noise in lung tissue with AIDR-3D images when compared to reconstructions without AIDR-3D (p = 0.005) and no significant change in the radiodensity (p = 1; mean difference <6 HU). Mean perfusion values increased significantly at lower tube currents (25 and 12.5 mAs), compared to 50 mAs (p = 0.005). This effect was significantly greater in larger patients compared to thin patients. CONCLUSION: AIDR-3D produced significantly lower noise images than FBP-based algorithms and maintained consistent noise levels in lung at 12.5 mAs, indicating this algorithm is suitable for reduced dose lung perfusion imaging. Iterative reconstruction allows significant radiation dose reduction of up to fourfold in smaller patients, and up to twofold in the medium/large size patients. The increase in perfusion values at 25% simulated tube currents is attributed to attenuation bias.

Sleep-disordered breathing and idiopathic pulmonary fibrosis--is there an association?

BACKGROUND: Very little data exists on the prevalence and impact of sleep-disordered breathing (SDB) in patients with idiopathic pulmonary fibrosis (IPF). The aim of this study was to examine the impact of SDB on newly diagnosed IPF patients and explore associations with lung function parameters. METHODS: From 2005 to 2009, a cohort of 27 newly diagnosed patients with IPF underwent unattended polygraphy. All patients were diagnosed according to ATS 2000 diagnostic criteria and were not on supplemental oxygen or other treatment. Standard statistical analysis was undertaken using SPSS v. 19 (IBM). RESULTS: Of the 27 patients, 19 were men. There was no correlation at baseline of apnoea + hypopnoea per time in bed (AH), oxygen desaturation index, or 4% desaturations with any lung function variables, age, or body mass index. Six patients had significant SDB (AH >20). Two patients were started on CPAP following polygraphy. No variables from the original sleep studies at baseline predicted eventual long-term oxygen therapy (LTOT) use. At 5-year follow-up, 18 of 27 patients had died (67%). Cox regression analysis showed no association of time spent at SpO2 <90% on baseline polygraphy with survival (p = 0.39). There was no association with survival for AH >20 (p = 0.4) or LTOT use (p = 0.19). CONCLUSION: Our results do not support the contention that nocturnal upper airway obstruction in steroid-free patients with IPF is a common problem or correlated with lung function. In this cohort of patients, there was no evidence that significant SDB at baseline was a predictor of survival.

Six cases of silicosis: implications for health surveillance of stonemasons.

BACKGROUND: Silicosis is one of the oldest occupational lung diseases, but it continues to cause significant morbidity and mortality worldwide. AIMS: To report cases of silicosis presenting to two specialist respiratory clinics. METHODS: A retrospective analysis of prospectively collected data of cases of silicosis in workers referred to specialist respiratory clinics. RESULTS: Over the course of 6 years, six cases were identified. The patients were all male with an age range between 24 and 39 years. The duration of silica exposure ranged between 7 and 20 years (mean 13 years). Four cases were entirely asymptomatic at presentation, and two cases described minimal shortness of breath on exertion. Pulmonary function tests were normal in three cases, and a mild restrictive ventilatory defect was documented in the other cases. All had a low apparent predicted probability of pneumoconiosis based on health questionnaires, spirometry and duration of silica exposure. The initial chest X-ray was abnormal in all six cases with radiological evidence of silicosis (International Labour Office profusion category ≥1/1) on imaging, and all had evidence of silicosis on high-resolution computed tomography (HRCT). Three patients had already progressed to progressive massive fibrosis on HRCT scanning at the time of referral to specialist respiratory services. CONCLUSIONS: The appearances of these six cases of silicosis in young, asymptomatic construction workers emphasizes the importance of enforcing effective exposure control and comprehensive surveillance programmes. Our observations highlight the importance of having a low threshold for early radiological screening to promote early and effective detection of this disease.

Downregulation of Mcl-1 has anti-inflammatory pro-resolution effects and enhances bacterial clearance from the lung.

Phagocytes not only coordinate acute inflammation and host defense at mucosal sites, but also contribute to tissue damage. Respiratory infection causes a globally significant disease burden and frequently progresses to acute respiratory distress syndrome, a devastating inflammatory condition characterized by neutrophil recruitment and accumulation of protein-rich edema fluid causing impaired lung function. We hypothesized that targeting the intracellular protein myeloid cell leukemia 1 (Mcl-1) by a cyclin-dependent kinase inhibitor (AT7519) or a flavone (wogonin) would accelerate neutrophil apoptosis and resolution of established inflammation, but without detriment to bacterial clearance. Mcl-1 loss induced human neutrophil apoptosis, but did not induce macrophage apoptosis nor impair phagocytosis of apoptotic neutrophils. Neutrophil-dominant inflammation was modelled in mice by either endotoxin or bacteria (Escherichia coli). Downregulating inflammatory cell Mcl-1 had anti-inflammatory, pro-resolution effects, shortening the resolution interval (Ri) from 19 to 7 h and improved organ dysfunction with enhanced alveolar-capillary barrier integrity. Conversely, attenuating drug-induced Mcl-1 downregulation inhibited neutrophil apoptosis and delayed resolution of endotoxin-mediated lung inflammation. Importantly, manipulating lung inflammatory cell Mcl-1 also accelerated resolution of bacterial infection (Ri; 50 to 16 h) concurrent with enhanced bacterial clearance. Therefore, manipulating inflammatory cell Mcl-1 accelerates inflammation resolution without detriment to host defense against bacteria, and represents a target for treating infection-associated inflammation.

The presence and regulation of connective tissue growth factor in the human endometrium.

BACKGROUND: The human endometrium efficiently repairs each month after menstruation. The mechanisms involved in this repair process remain undefined. Aberrations in endometrial repair may lead to the common disorder of heavy menstrual bleeding. We hypothesized that connective tissue growth factor (CTGF) is increased at the time of endometrial repair post-menses and that this increase is regulated by prostaglandins (PGs) and hypoxic conditions present during menstruation. METHODS AND RESULTS: Examination of 41 endometrial biopsies from 5 stages of the menstrual cycle revealed maximal CTGF mRNA expression (using quantitative RT-PCR) at menstruation and peak protein levels during the proliferative phase. CTGF was immunolocalized to epithelial and stromal cells, with intense staining of occasional stromal cells during the proliferative phase. Dual immunohistochemistry identified these cells as macrophages. Treatment of endometrial epithelial cells with 100 nM PGE(2), PGF(2α) or hypoxia (0.5% O(2)) revealed a significant increase in CTGF mRNA expression (P < 0.01 for all, versus vehicle control). Cells treated simultaneously with PGE(2) and hypoxia revealed a synergistic increase in CTGF expression (P < 0.05 versus PGE(2) or hypoxia alone) and maximal secreted CTGF protein levels (P < 0.05 versus control). CONCLUSIONS: CTGF is increased in the human endometrium at the time of endometrial repair post-menses. The increase in CTGF may be mediated by PG production and the transient hypoxic episode observed in the endometrium at menstruation.

The regulation of interleukin-8 by hypoxia in human macrophages--a potential role in the pathogenesis of the acute respiratory distress syndrome (ARDS).

BACKGROUND: The acute respiratory distress syndrome (ARDS) represents a form of severe acute inflammatory lung disease. We have previously demonstrated significantly raised interleukin-8 (IL-8) levels in the lungs of at-risk patients that progress to ARDS, and identified the alveolar macrophage as an important source of this chemokine. We wished to extend this study in a well-defined group of patients with major trauma, and to investigate potential mechanisms for rapid intrapulmonary IL-8 generation. MATERIALS AND METHODS: Patients with major trauma underwent bronchoalveolar lavage (BAL) and IL-8 levels were measured in BAL fluid by ELISA. Human macrophages were derived from peripheral blood monocytes from healthy volunteers. Rabbit alveolar macrophages were obtained from ex-vivo lavage of healthy rabbit lungs. Macrophages were culture under normoxic or hypoxic (PO2 26 mmHg) conditions. IL-8 and other proinflammatory mediator expression was measured by ELISA, northern blotting or multi-probe RNase protection assay. RESULTS: In patients with major trauma, IL-8 levels were significantly higher in patients that progressed to ARDS compared to those that did not (n = 56, P = 0.0001). High IL-8 levels negatively correlated with PaO2/FiO2 (r = -0.56, P < 0.001). In human monocyte derived macrophages hypoxia rapidly upregulated IL-8 protein (within 2 hours) and mRNA expression (within 30 mins). Acute hypoxia also increased rabbit alveolar macrophage IL-8 expression. Hypoxia increased DNA binding activity of AP-1 and C/EBP but not NF-kappaB. Hypoxia induced HIF-1 expression, but cobaltous ions and desferrioxamine did not mimic hypoxic IL-8 induction. Hypoxia downregulated a range of other proinflammatory mediators, including MCP-1 and TNF-alpha. Both the pattern of cytokine expression and transcription factor activation by hypoxia was different to that seen with endotoxin. CONCLUSIONS: Rapidly raised intrapulmonary IL-8 levels are associated with ARDS progression in patients with major trauma. Acute hypoxia, a clinically relevant stimulus, rapidly and selectively upregulates IL-8 in macrophages associated with a novel pattern of transcription factor activation. Acute hypoxia may represent one of potentially several proinflammatory stimuli responsible for rapid intrapulmonary IL-8 generation in patients at-risk of ARDS.

Elevated levels of interleukin-8 in donor lungs is associated with early graft failure after lung transplantation.

Increased levels of the neutrophil chemokine interleukin (IL)-8 in the lungs of severe trauma patients can predict subsequent development of acute respiratory distress syndrome. Because the lungs of brain-dead organ donors can contain high levels of IL-8, we hypothesized that this may predispose to early graft failure in the recipient after lung transplantation. Twenty-six organ donors prospectively satisfying clinical criteria for lung donation underwent bronchoalveolar lavage and lung biopsy to determine the effect of neutrophil infiltration and IL-8 expression in the donor lung on graft function and survival in 26 respective recipients after lung transplantation. Nine recipients developed severe graft dysfunction, of whom six subsequently died (median survival: 24 d [range: 5 to 39 d]); all others survived beyond 6 mo. The IL-8 signal in the donor lung correlated with the percent neutrophils in bronchoalveolar lavage fluid (BALF) before implantation (42.4 +/- 7.24 [mean +/- SE]%, p = 0.03) and with the degree of impairment in graft oxygenation after implantation (p = 0.01). An increased level of IL-8 in the donor BALF was associated with the development of severe early graft dysfunction (p = 0.027) and with early recipient mortality (p = 0.0034). Use of donor lungs with high IL-8 levels is associated with a poor prognosis after lung transplantation. Attenuating the donor's inflammatory response before organ retrieval may improve early outcome after lung transplantation, and help maximize lung use from the existing donor pool.

Regulation of lipopolysaccharide-mediated interleukin-1beta release by N-acetylcysteine in THP-1 cells.

Increased levels of inflammatory cytokines such as interleukin (IL)-1 and IL-8 occur in the bronchoalveolar lavage fluid in various lung diseases. Cytokine gene expression is controlled by transcription factors such as nuclear factor-kappaB (NF-kappaB) which can be activated by a number of stimuli including the oxidants prevent. It was hypothesized that lipopolysaccharide (LPS)-induced IL-1beta secretion may be modulated by the intracellular thiol redox status of the cells. The effect of the antioxidant compound, N-acetyl-L-cysteine (NAC), on IL-1beta release and regulation of NF-kappaB in a human myelo-monocytic cell line (THP-1) differentiated into macrophages was studied. LPS (10 microg x mL(-1)) increased IL-1beta release at 24 h compared to control levels (p<0.001). NAC (5 mM) also enhanced LPS-induced IL-1beta release from THP-1 cells (p<0.001). In addition, treatment of cells with cycloheximide, an inhibitor of protein synthesis, inhibited the NAC-mediated IL-1beta release. Under the same conditions, NF-kappaB binding was activated by LPS and NAC increased this LPS-mediated effect. Western blot analysis revealed that NAC treatment leads to an increase in p50 and p65 protein synthesis. These data indicate that N-acetyl-L-cysteine modulates interleukin-1kappa release by increasing levels of the homo- and heterodimeric forms of nuclear factor-kappaB.

Human circulating eosinophils secrete macrophage migration inhibitory factor (MIF). Potential role in asthma.

Macrophage migration inhibitory factor (MIF) is a potent proinflammatory mediator that has been shown to potentiate lethal endotoxemia and to play a potentially important regulatory role in human acute respiratory distress syndrome (ARDS). We have investigated whether eosinophils are an important source of MIF and whether MIF may be involved in the pathophysiology of asthma. Unstimulated human circulating eosinophils were found to contain preformed MIF. Stimulation of human eosinophils with phorbol myristate acetate in vitro yielded significant release of MIF protein. For example, eosinophils stimulated with phorbol myristate acetate (100 nM, 8 h, 37 degreesC) released 1,539+/-435 pg/10(6) cells of MIF, whereas unstimulated cells released barely detectable levels (< 142 pg/10(6) cells, mean+/-SEM, n = 8). This stimulated release was shown to be (a) concentration- and time-dependent, (b) partially blocked by the protein synthesis inhibitor cycloheximide, and (c) significantly inhibited by the protein kinase C inhibitor Ro-31,8220. In addition, we show that the physiological stimuli C5a and IL-5 also cause significant MIF release. Furthermore, bronchoalveolar lavage fluid obtained from asthmatic patients contains significantly elevated levels of MIF as compared to nonatopic normal volunteers (asthmatic, 797.5+/-92 pg/ml; controls, 274+/-91 pg/ml). These results highlight the potential importance of MIF in asthma and other eosinophil-dependent inflammatory disorders.

Impact of management guidelines on the outcome of severe community acquired pneumonia.

BACKGROUND: Ten years ago we published a study of 50 adults with severe community acquired pneumonia admitted to our intensive care unit and subsequently introduced guidelines for the management of severe community acquired pneumonia which are largely in accordance with those of the British Thoracic Society. The results of a follow up study are now reported in order to assess their impact on the outcome of this disease. METHODS: Fifty seven cases of severe community acquired pneumonia admitted to our ICU between 1984 and 1993 were studied. Causal pathogens, clinical and laboratory features of severity, antibiotic therapy and mortality were studied and, where possible, compared with results from the previous study. RESULTS: Streptococcus pneumoniae, Legionella pneumophila and Staphylococcus aureus were the most frequent causes of severe community acquired pneumonia, as in the previous study. The intensity of microbial investigation has increased, particularly with regard to pneumococcal and Legionella antigen testing, the latter allowing earlier diagnosis of Legionella infection than previously. In spite of this, no pathogen was identified in 33% of cases compared with 18% previously. Indices of severity of illness were widely recognised, and a decrease in unplanned transfers to the ICU following "unexpected" cardiorespiratory arrest from 25% to 7% (p < 0.02) was found. Antibiotic therapy largely reflected guideline recommendations with 98% receiving a beta-lactam agent and 91% erythromycin. The overall mortality was 58% compared with 54% previously. CONCLUSIONS: Management guidelines for severe community acquired pneumonia have been widely adopted but without a reduction in mortality in our hospital. Factors other than early diagnosis, appropriate antibiotics, or prompt ICU transfer may influence the outcome in severe community acquired pneumonia.

Central and peripheral SEP defects in neurologically symptomatic and asymptomatic subjects with low vitamin B12 levels.

Somatosensory evoked potentials (SEPs) following median nerve stimulation were abnormal in 7 patients with sensory impairment due to vitamin B12 deficiency. Extensor plantar reflexes indicated a central sensory pathway lesion in 4 cases and absent tendon jerks suggested peripheral neuropathy in 4, but median nerve SEPs indicated a predominantly central lesion without marked peripheral nerve involvement in 6 and an axonal neuropathy without CNS involvement in 1. The latter had evidence of central slowing of conduction in SEPs following posterior tibial nerve stimulation. Consequently, it is suggested that the brunt of sensory pathway involvement usually falls on the CNS, although peripheral neuropathy may occur as the major abnormality in some cases. In 2 patients SEPs showed a marked improvement following treatment with vitamin B12 injections, one consistent with restored central conduction and the other with recovery from peripheral neuropathy. No peripheral or central SEP abnormalities were seen in 18 dairy-produce eating vegetarians with low vitamin B12 levels, although 6 reported mild sensory symptoms suggestive of peripheral neuropathy and 3 had corroborative clinical signs.