Clinical Evaluation of the XDR-LFC Assay for the Molecular Detection of Isoniazid, Rifampin, Fluoroquinolone, Kanamycin, Capreomycin, and Amikacin Drug Resistance in a Prospective Cohort.

While the goal of universal drug susceptibility testing has been a key component of the WHO End TB Strategy, in practice, this remains inaccessible to many. Rapid molecular tests for tuberculosis (TB) and antituberculosis drug resistance could significantly improve access to testing. In this study, we evaluated the accuracy of the Akonni Biosystems XDR-TB (extensively drug-resistant TB) TruArray and lateral-flow-cell (XDR-LFC) assay (Akonni Biosystems, Inc., Frederick, MD, USA), a novel assay that detects mutations in seven genes associated with resistance to antituberculosis drugs: katG, the inhA promoter, and the ahpC promoter for isoniazid; rpoB for rifampin; gyrA for fluoroquinolones; rrs and the eis promoter for kanamycin; and rrs for capreomycin and amikacin. We evaluated assay performance using direct sputum samples from 566 participants recruited in a prospective cohort in Moldova over 2 years. The sensitivity and specificity against the phenotypic reference were both 100% for isoniazid, 99.2% and 97.9% for rifampin, 84.8% and 99.1% for fluoroquinolones, 87.0% and 84.1% for kanamycin, 54.3% and 100% for capreomycin, and 79.2% and 100% for amikacin, respectively. Whole-genome sequencing data for a subsample of 272 isolates showed 95 to 99% concordance with the XDR-LFC-reported suspected mutations. The XDR-LFC assay demonstrated a high level of accuracy for multiple drugs and met the WHO's minimum target product profile criteria for isoniazid and rifampin, while the sensitivity for fluoroquinolones and amikacin fell below target thresholds, likely due to the absence of a gyrB target in the assay. With optimization, the XDR-LFC shows promise as a novel near-patient technology to rapidly diagnose drug-resistant tuberculosis.

Laboratory Evaluation of a Lateral-Flow Cell for Molecular Detection of First-Line and Second-Line Antituberculosis Drug Resistance.

Despite the WHO's call for universal drug susceptibility testing for all patients being evaluated for tuberculosis (TB), a lack of rapid diagnostic tests which can fully describe TB resistance patterns is a major challenge in ensuring that all persons diagnosed with drug-resistant TB are started on an appropriate treatment regime. We evaluated the accuracy of the Akonni Biosystems XDR-TB TruArray and lateral-flow cell (XDR-LFC), a novel multiplex assay to simultaneously detect mutations across seven genes that confer resistance to both first- and second-line anti-TB drugs. The XDR-LFC includes 271 discrete three-dimensional gel elements with target-specific probes for identifying mutations in katG, inhA promoter, and ahpC promoter (isoniazid), rpoB (rifampin), gyrA (fluoroquinolones), rrs and eis promoter (kanamycin), and rrs (capreomycin and amikacin). We evaluated XDR-LFC performance with 87 phenotypically and genotypically characterized clinical Mycobacterium tuberculosis isolates. The overall assay levels of accuracy for mutation detection in specific genes were 98.6% for eis promoter and 100.0% for the genes katG, inhA promoter, ahpC promoter, rpoB, gyrA, and rrs The sensitivity and specificity against phenotypic reference were 100% and 100% for isoniazid, 98.4% and 50% for rifampin (specificity increased to 100% once the strains with documented low-level resistance mutations in rpoB were excluded), 96.2% and 100% for fluoroquinolones, 92.6% and 100% for kanamycin, 93.9% and 97.4% for capreomycin, and 80% and 100% for amikacin. The XDR-LFC solution appears to be a promising new tool for accurate detection of resistance to both first- and second-line anti-TB drugs.

Fibrinogen and the prediction of residual obstruction manifested after pulmonary embolism treatment.

Residual pulmonary vascular obstruction (RPVO) and chronic thromboembolic pulmonary hypertension (CTEPH) are both long-term complications of acute pulmonary embolism, but it is unknown whether RPVO can be predicted by variants of fibrinogen associated with CTEPH.We used the Akaike information criterion to select the best predictive models for RPVO in two prospectively followed cohorts of acute pulmonary embolism patients, using as candidate variables the extent of the initial obstruction, clinical characteristics and fibrinogen-related data. We measured the selected models' goodness of fit by analysis of deviance and compared models using the Chi-squared test.RPVO occurred in 29 (28.4%) out of 102 subjects in the first cohort and 46 (25.3%) out of 182 subjects in the second. The best-fit predictive model derived in the first cohort (p=0.0002) and validated in the second cohort (p=0.0005) implicated fibrinogen Bß-chain monosialylation in the development of RPVO. When the derivation procedure excluded clinical characteristics, fibrinogen Bß-chain monosialylation remained a predictor of RPVO in the best-fit predictive model (p=0.00003). Excluding fibrinogen characteristics worsened the predictive model (p=0.03).Fibrinogen Bß-chain monosialylation, a common structural attribute of fibrin, helped predict RPVO after acute pulmonary embolism. Fibrin structure may contribute to the risk of developing RPVO.

Structural insights into fibrinogen dynamics using amide hydrogen/deuterium exchange mass spectrometry.

We determined the amide hydrogen/deuterium exchange profile of native human fibrinogen under physiologic conditions. After optimization of the quench and proteolysis conditions, more than 1,200 peptides were identified by mass spectrometry, spanning more than 90% of the constituent Aα, Bß, and γ chain amino acid sequences. The compact central and distal globular regions of fibrinogen were well protected from deuterium exchange, with the exception of the unfolded amino-terminal segments of the Aα and Bß chains extending from the central region, and the short γ chain "tail" extending from each distal globular region. The triple-helical coiled-coil regions, which bridge the central region to each distal region, were also well protected with the exception of a moderately fast-exchanging area in the middle of each coiled-coil adjacent to the γ chain carbohydrate attachment site. These dynamic regions appear to provide flexibility to the fibrinogen molecule. The γ chain "out loop" contained within each coiled-coil also exchanged rapidly. The αC domain (Aα 392-610) exchanged rapidly, with the exception of a short segment sandwiched between a conserved disulfide linkage in the N-terminal αC subdomain. This latter finding is consistent with a mostly disordered structure for the αC domain in native fibrinogen. Analysis of the dysfibrinogen Bß 235 Pro/Leu, which exhibits abnormal fibrin structure, revealed enhanced deuterium exchange surrounding the Pro/Leu substitution site as well as in the vicinity of the high affinity calcium binding site and the A knob polymerization pocket within the γC domain. The implication of these changes with respect to fibrin structure is discussed.

Detecting rifampin and isoniazid resistance in Mycobacterium tuberculosis direct from patient sputum using an automated integrated system.

While there has been progress in detection of drug resistant tuberculosis globally, WHO estimates only about half of the patients with bacteriologically confirmed tuberculosis were tested for rifampicin resistance over the past two years. To close this drug resistance diagnostic gap, an expansion of testing for rifampicin and isoniazid resistance is critically needed. The Akonni Biosystem Integrated System combines DNA extraction and a Lab-on-a-Film assembly (LFA) to perform rapid probe and PCR-based detection of resistance associated mutations to first-line anti-tuberculosis drugs. Using raw sputum samples from 25 tuberculosis patients at risk for drug resistance, we conducted a proof-of-concept study of the Integrated System with an MDR-TB assay. Performance of the Integrated System was compared to liquid Mycobacteria Growth Indicator Tube (MGIT) culture reference phenotypes using 2012 WHO endorsed critical concentrations for rifampicin and isoniazid. The overall percent agreement for rifampicin and isoniazid was 91.7% and 100% respectively, with agreement for rifampicin increasing to 95.7% after low-level resistance mutations in rpoB were excluded. The Integrated System, combining DNA extraction and LFA amplification, is a promising new tool for detection of both rifampicin and isoniazid using liquefied raw sputum.

High prevalence of dysfibrinogenemia among patients with chronic thromboembolic pulmonary hypertension.

The mechanism by which chronic thromboembolic pulmonary hypertension (CTEPH) develops after acute pulmonary thromboembolism is unknown. We previously reported that fibrin from CTEPH patients is relatively resistant to fibrinolysis in vitro. In the present study, we performed proteomic, genomic, and functional studies on fibrin(ogen) to investigate whether abnormal fibrin(ogen) might contribute to the pathogenesis of CTEPH. Reduced and denatured fibrinogen from 33 CTEPH patients was subjected to liquid chromatography-mass spectrometry analysis. Fibrinogen from 21 healthy controls was used to distinguish atypical from commonly occurring mass peaks. Atypical peaks were further investigated by targeted genomic DNA sequencing. Five fibrinogen variants with corresponding heterozygous gene mutations (dysfibrinogenemias) were observed in 5 of 33 CTEPH patients: Bbeta P235L/gamma R375W, Bbeta P235L/gamma Y114H, Bbeta P235L, Aalpha L69H, and Aalpha R554H (fibrinogens(San Diego I-V)). Bbeta P235L was found in 3 unrelated CTEPH patients. Functional analysis disclosed abnormalities in fibrin polymer structure and/or lysis with all CTEPH-associated mutations. These results suggest that, in some patients, differences in the molecular structure of fibrin may be implicated in the development of CTEPH after acute thromboembolism.

Rapid Detection of Extensively Drug-Resistant Tuberculosis in Clinical Samples Using a Novel Tabletop Platform: Protocol for a Prospective Clinical Study.

BACKGROUND: The lack of accurate and efficient diagnostic devices for extensively drug-resistant tuberculosis (XDR-TB) makes it a severe threat to global public health. A prospective clinical study in an intended-use cohort was designed to evaluate the Akonni Biosystems XDR-TB TruArray and lateral flow cell (XDR-LFC) to address this gap in tuberculosis diagnostics. OBJECTIVE: This paper presents the protocol for a study that aims to document the conceptualization and design of this evaluation method for early dissemination while data collection and analysis are ongoing. METHODS: The clinical study was conducted in three phases. The first phase was to observe changes in bacterial load and culture positivity in patient sputa over time and better understand the diversity of prospective clinical samples. The second phase was to prospectively collect clinical samples for sensitivity and specificity testing of the Akonni Biosystems XDR-LFC device. Lastly, the third phase was to explore the anti-TB drug concentrations in serum throughout the drug-resistant tuberculosis treatment. RESULTS: The methodology described includes the study design, laboratory sample handling, data collection, and the protection elements of human subjects of this clinical study to evaluate a potential new XDR-TB diagnostic device. A total of 664 participants were enrolled across the three phases. The implemented complex systems facilitated a thorough clinical data collection for an objective evaluation of the device. The study is closed to recruitment. The follow-up data collection and analysis are in progress. CONCLUSIONS: This paper outlined a prospective cohort study protocol to evaluate a rapid XDR-TB detection device, which may be informative for other researchers with similar goals. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/26748.

Antibiotics against Pseudomonas aeruginosa for COPD exacerbation in ICU: a 10-year retrospective study.

SUMMARY: Chronic obstructive pulmonary disease (COPD) is a frequent source of hospitalization. Antibiotics are largely prescribed during COPD exacerbation. Our hypothesis is that large broad-spectrum antibiotics are more and more frequently prescribed. Our results confirm this trend and highlight that the increase in large broad-spectrum use in COPD exacerbation is largely unexplained. BACKGROUND: Acute COPD exacerbation (AECOPD) is frequently due to respiratory tract infection, and the benefit of antipseudomonal antibiotics (APA) is still debated. Health care-associated pneumonia (HCAP) was defined in 2005 and requires broad-spectrum antibiotherapy. The main objectives are to describe the antibiotic use for AECOPD in intensive care unit and to identify factors associated with APA use and AECOPD prognosis. METHODS: We conducted a monocentric, retrospective study on all AECOPDs in the intensive care unit treated by antibiotics for respiratory tract infection. Treatment failure (TF) was defined by death, secondary need for mechanical ventilation, or secondary systemic steroid treatment. A multivariate analysis was used to assess factors associated with APA prescription and TF. RESULTS: From January 2000 to December 2011, 111 patients were included. Mean age was 69 years (±12), mean forced expiratory volume 38% of theoretic value (±13). Thirty-five (31%) patients were intubated, and 52 (47%) were treated with noninvasive ventilation. From 107 patients, 8 (7%) cases of Pseudomonas aeruginosa were documented. APAs were prescribed in 21% of patients before 2006 versus 57% after (P=0.001). TF prevalence was 31%. Risk factors for P. aeruginosa in COPD and HCAP diagnosis did not influence APA, whereas the post-2006 period was independently associated with APA prescription (odds ratio 6.2; 95% confidence interval 1.9-20.3; P=0.0013). APA did not improve TF (odds ratio 1.09; 95% confidence interval 0.37-3.2). CONCLUSION: HCAP guidelines were followed by an increase in APA use in AECOPD, without an improvement in prognosis. HCAP prevalence cannot account for the increasing APA trend. Time effect reveals a drift in practices. The microbiological effect of such a drift must be evaluated.

Urine and plasma levels of fibrinopeptide B in patients with deep vein thrombosis and pulmonary embolism.

BACKGROUND: Many patients with pulmonary thromboembolism remain undiagnosed, possibly because of the difficulty clinicians have in determining which patients merit work-up with accurate (but expensive) imaging techniques. OBJECTIVES: We present the first prospective clinical study of pulmonary embolism (PE) and deep venous thrombosis (DVT) detection using the FPBtot assay, which measures fibrinopeptide B and its first derivative, des-arginine fibrinopeptide B. METHODS: Twenty three patients with signs or symptoms of PE or DVT were enrolled in the study prior to the performance of definitive testing. Using a novel immunoassay, FPBtot levels were measured in urine and plasma samples from patients as well as from healthy controls. Urine and plasma FPBtot levels were compared to the diagnostic results, as blindly adjudicated by one of the investigators. Patients were excluded if they withdrew (n =1), had inconclusive diagnostic testing (n = 7), or did not give samples (n = 2 for urine, n = 3 for plasma). RESULTS: The mean FPBtot concentration in the urine of the 'DVT/PE positive' group was 78.4 +/- 35.2 ng/ml and 2.7 +/- 1.9 ng/ml in the 'DVT/PE negative' group (p = 0.03). The urine FPB(tot) concentrations in the 'DVT/PE negative' group were not significantly different from those in the healthy control group (2.2 +/- 0.4 ng/ml, p = 0.40). The area under the ROC curve for urine FPB(tot) concentrations was 97.3 +/- 3.8%, suggesting a high degree of diagnostic accuracy. Plasma FPB(tot) concentrations were not significantly different between groups. CONCLUSIONS: Urine FPBtot levels may help detect patients with PE and DVT.

Improved imaging of deep venous thrombi during anticoagulation using radiolabelled anti-D-dimer antibodies.

OBJECTIVES: Radiolabelled anti-fibrin antibodies have not yet enabled reliable and practical diagnosis of venous thromboembolism. However, previous unsuccessful clinical trials were performed with anti-fibrin beta-chain antibodies that do not optimally bind to thrombi during anticoagulation. The current experiments were performed to determine if radiolabelled anti-D-dimer antibodies more reliably allowed nuclear medicine imaging of deep venous thrombi during anticoagulation than anti-beta-chain antibodies. METHODS: Dogs with pre-existing unilateral femoral vein thrombi were anticoagulated with heparin (300 units.kg (-1) bolus followed by 90 units.kg(-1).h(-1) continuous infusion). They were then allocated to receive one of three (111)In labelled antibodies: anti-D-dimer, anti-beta or a non-specific control antibody. Gamma scans of the legs were performed at regular intervals for 24 h. Scans were interpreted in a blinded fashion and the number of gamma counts from the femoral area on the thrombosed side was compared to the contralateral side. Clot/blood isotope density ratios were determined post-mortem. RESULTS: Leg thrombi were 100% detectable in the anti-D-dimer group, but only 60% detectable in the anti-beta group. Mean +/- SD relative counts in the thrombosed femoral area was 137 +/- 10% compared to the contralateral side in the anti-D-dimer group, but only 116 +/- 20% in the anti-beta group. The clot/blood ratio was 24.5 +/- 2.8 in the anti-D-dimer group, but only 7.8 +/- 2.0 in the anti-beta group. CONCLUSIONS: In labelled anti-D-dimer provides superior nuclear medicine images of thrombi during intensive anticoagulation compared to anti-beta. Clinical results with radiolabelled anti-D-dimer may be more promising than those previously observed with other anti-fibrin antibodies.

Chronic thromboembolic pulmonary hypertension-associated dysfibrinogenemias exhibit disorganized fibrin structure.

INTRODUCTION: Mechanisms contributing to the pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) are poorly understood. This disorder is characterized by incomplete resolution of pulmonary perfusion defects resulting from acute venous thromboembolism. We previously identified several dysfibrinogenemias in some patients with CTEPH. The purpose of this study was to determine whether fibrin clot architecture might be implicated in the thrombolytic resistance in patients with these CTEPH-associated dysfibrinogenemias. MATERIALS AND METHODS: Purified fibrinogen from patients and healthy controls was clotted with thrombin in the presence of calcium. Clot turbidity, porosity, and susceptibility to fibrinolysis were evaluated by spectrophotometric and permeation analyses. Fibrin network structure was assessed by laser-scanning confocal microscopy. RESULTS: Compared to normal fibrinogen, CTEPH-associated dysfibrinogenemias exhibited low clot turbidity, decreased porosity, and fibrinolytic resistance. In addition, the dysfibrinogenemias exhibited a more disorganized fibrin network structure characterized by thinner fibers, greater network dispersal and more extensive fiber branching. CONCLUSIONS: Abnormal clot architecture and fibrinolytic resistance may contribute to incomplete clot resolution following acute venous thromboembolism in patients with CTEPH-associated dyfibrinogenemia.

Chronic exposure to fibrin and fibrinogen differentially regulates intracellular Ca2+ in human pulmonary arterial smooth muscle and endothelial cells.

Acute pulmonary embolism occurs in more than half a million people a year in the United States. Chronic thromboembolic pulmonary hypertension (CTEPH) develops in approximately 4% of these patients due to unresolved thromboemboli. CTEPH is thus a relatively common, progressive, and potentially fatal disease. One currently proposed theory for the poor resolution advocates that modification of fibrinogen in CTEPH patients causes resistance of emboli to fibrinolysis. The current study investigated the regulation of cytosolic Ca(2+) ([Ca(2+)](cyt)), central to the control of cell migration, proliferation, and contraction, by chronic exposure of pulmonary artery smooth muscle (PASMC) and endothelial (PAEC) cells to fibrinogen and fibrin. Basal [Ca(2+)](cyt) was substantially elevated in PAEC after culture on fibrinogen, fibrin, and thrombin and in PASMC on fibrinogen and fibrin. In PAEC, fibrinogen significantly decreased the peak [Ca(2+)](cyt) transient (P <0.001) without a change in the transient peak width (at 50% of the peak height). This response was independent of effects on the proteinase-activated receptor (PAR) 1. Furthermore, chronic exposure to thrombin, an activator of PAR, significantly reduced the peak agonist-induced Ca(2+) release in PAEC, but increased it in PASMC. The recovery rate of the agonist-induced [Ca(2+)](cyt) transients decelerated in PASMC chronically exposed to fibrin; a small increase of the peak Ca(2+) was also observed. Substantial augmentation of PASMC (but not PAEC) proliferation was observed in response to chronic fibrin exposure. In conclusion, chronic exposure to fibrinogen, fibrin, and thrombin caused differential changes in [Ca(2+)](cyt) in PAEC and PASMC. Such changes in [Ca(2+)](cyt) may contribute to vascular changes in patients who have CTEPH where the pulmonary vasculature is persistently exposed to thromboemboli.

Fibrin derived from patients with chronic thromboembolic pulmonary hypertension is resistant to lysis.

RATIONALE: Although acute pulmonary embolism is epidemiologically associated with chronic thromboembolic pulmonary hypertension, the factors responsible for resistance to thrombolysis and a shift toward vascular remodeling within the pulmonary arteries of patients with chronic thromboembolic pulmonary hypertension are unknown. OBJECTIVE: Determine whether fibrin from patients is more resistant to plasmin-mediated lysis than fibrin from healthy control subjects. METHODS: Fibrinogen purified from patients and control subjects was used to prepare fibrin clots, which were subsequently digested with plasmin for various periods of time. The degradation of the alpha-, beta-, and gamma-chains of fibrin and the appearance of peptide fragments over time were assessed by polyacrylamide gel electrophoresis and Western blotting. MEASUREMENTS AND MAIN RESULTS: Densitometry of Coomassie-stained gels revealed significantly slower cleavage of all three polypeptide chains of fibrin from patients compared with control subjects (p < 0.05). In particular, release of N-terminal fragments from the beta-chain of fibrin, which promote cell signaling, cell migration, and angiogenesis, was retarded in patients compared with control subjects (p < 0.01). CONCLUSIONS: The relative resistance of patient fibrin to plasmin-mediated lysis may be due to alterations in fibrin(ogen) structure affecting accessibility to plasmin cleavage sites. The persistence of structural motifs of fibrin, such as the beta-chain N-terminus, within the pulmonary vasculature could promote the transition from acute thromboemboli into chronic obstructive vascular scars.

Embolization itself stimulates thrombus propagation in pulmonary embolism.

The role of active thrombosis in the pathophysiology of pulmonary embolism is unclear. We tested the hypothesis that venous thrombi significantly increase their thrombotic activity once they embolize into the high-flow circulation of the pulmonary arteries. Thrombotic activity was measured using an immunoassay that measures both fibrinopeptide B (FPB) as well as its most abundant metabolite des-arginine FPB. Thrombi were formed in the femoral veins of adult dogs. In one group, the thrombi were embolized without anticoagulation. In the second group, heparin (300 U/kg bolus, then 90 U x kg(-1) x h(-1) infusion) was administered before embolization to prevent subsequent thrombotic activity. Plasma FPB concentrations were significantly suppressed in the heparinized group relative to the nonheparinized group for 1 h postembolization (P = 0.038). We conclude that pulmonary embolization itself causes preexisting venous thrombi to greatly intensify their thrombotic activity and that embolization-associated thrombus propagation can be prevented by heparin.

Single photon emission computed tomography of pulmonary emboli and venous thrombi using anti-D-dimer.

Previous attempts to diagnose thromboemboli using radiolabeled antibodies and nuclear medicine imaging have been disappointing. We present the results of experiments with intravenous technetium-99m-labeled deimmunized antifibrin Fab' fragments to diagnose thromboemboli using single photon emission computed tomography (SPECT), a highly sensitive scintigraphic imaging technique. Pulmonary emboli (PEs) and lower extremity deep vein thrombi (DVTs) were formed in five dogs, then technetium-99m-labeled Fab' ( approximately 400 mg, approximately 260 MBq) were injected via forelimb veins. Thoracic and lower extremity SPECT scans were performed at 2-hour intervals after antibody infusion to visualize the thromboemboli. Four hours after antibody infusion, all PEs and DVTs of mass 0.4 g or greater were clearly visualized on SPECT scans as 'hot spots' within the lungs and legs, respectively. PEs (0.48 +/- 0.09 g) were intensely radiolabeled, yielding clot/blood radioactivity ratios of 22.8 +/- 5.6. DVTs (0.45 +/- 0.31 g) also had high clot/blood ratios (11.7 +/- 2.6). Infusion of these radiolabeled antibody fragments, combined with SPECT, produces clear images of PEs and DVTs within a clinically feasible time frame. The technique reliably identified even peripheral thromboemboli of relatively small size, which are difficult to diagnose with currently available imaging techniques, and may enable imaging of PEs, DVTs, or both in the same patient.