Safety and efficacy of neurostimulation with a miniaturised vagus nerve stimulation device in patients with multidrug-refractory rheumatoid arthritis: a two-stage multicentre, randomised pilot study.

Background The inflammatory reflex plays a role in regulating innate and adaptive immunity by modulating cellular and molecular inflammatory pathways. The vagus nerve is a major constituent of the inflammatory reflex and studies have shown that the reflex can be activated by electrical stimulation of the vagus nerve. In this first in-human pilot study, we assessed the safety and efficacy of a novel miniaturised vagus nerve stimulation (VNS) device for the treatment of multidrug-refractory rheumatoid arthritis. METHODS: Participants with moderately to severely active rheumatoid arthritis and prior insufficient response to two or more biological disease-modifying anti-rheumatic drugs or Janus kinase inhibitors with at least two different modes of action were enrolled in a two-stage study done at five clinical research sites in the USA. Stage 1 was open label; participants were implanted with a miniaturised VNS device, which was activated for 1 min once a day. In stage 2, participants were randomly assigned (1:1:1) to receive active stimulation (1 min once a day or 1 min four times a day) or sham stimulation (device implanted but not activated), with the sites and participants masked to treatment assignment. The primary outcome was incidence of treatment-emergent adverse events. Clinical efficacy was assessed as a key secondary outcome. The study was registered with ClinicalTrials.gov, NCT03437473. FINDINGS: 14 patients were enrolled between March 13 and Aug 8, 2018. Three patients received stimulation in stage 1 and, following safety review board approval, the remaining 11 patients were implanted during stage 2 and randomly assigned to receive 1 min of stimulation once daily (n=3), 1 min of stimulation four times daily (n=4), or no stimulation (n=4) for 12 weeks. There were no device-related or treatment-related serious adverse events. Surgery-related adverse events were Horner's syndrome and vocal cord paralysis (in one patient each), which resolved without clinically significant sequelae. No deaths were recorded. INTERPRETATION: VNS with a miniaturised neurostimulator was safe and well tolerated and reduced signs and symptoms of rheumatoid arthritis in patients with multidrug-refractory disease. These results support further evaluation in a larger randomised sham-controlled study. FUNDING: SetPoint Medical.

Molecular and Growth-Based Drug Susceptibility Testing of Mycobacterium tuberculosis Complex for Ethambutol Resistance in the United States.

Ethambutol (EMB) is used as a part of drug regimens for treatment of tuberculosis (TB). Susceptibility of Mycobacterium tuberculosis complex (MTBC) isolates to EMB can be discerned by DNA sequencing to detect mutations in the embB gene associated with resistance. US Public Health Laboratories (PHL) primarily use growth-based drug susceptibility test (DST) methods to determine EMB resistance. The Centers for Disease Control and Prevention (CDC) provides a service for molecular detection of drug resistance (MDDR) by DNA sequencing and concurrent growth-based DST using agar proportion. PHL and CDC test results were compared for 211 MTBC samples submitted to CDC from September 2009 through February 2011. Concordance between growth-based DST results from PHL and CDC was 88.2%. A growth-based comparison of 39 samples, where an embB mutation associated with EMB resistance was detected, revealed a higher percentage of EMB resistance by CDC (84.6%) than by PHL (59.0%) which was significant (P value = 0.002). Discordance between all growth-based test results from PHL and CDC was also significant (P value = 0.003). Most discordance was linked to false susceptibility using the BACTEC™ MGIT™ 960 (MGIT) growth-based system. Our analysis supports coalescing growth-based and molecular results for an informed interpretation of potential EMB resistance.

Concordance between molecular and phenotypic testing of Mycobacterium tuberculosis complex isolates for resistance to rifampin and isoniazid in the United States.

Multidrug-resistant (MDR) isolates of Mycobacterium tuberculosis complex (MTBC) are defined by resistance to at least rifampin (RMP) and isoniazid (INH). Rapid and accurate detection of multidrug resistance is essential for effective treatment and interruption of disease transmission of tuberculosis (TB). Overdiagnosis of MDR TB may result in treatment with second-line drugs that are more costly, less effective, and more poorly tolerated than first-line drugs. CDC offers rapid confirmation of MDR TB by the molecular detection of drug resistance (MDDR) for mutations associated with resistance to RMP and INH along with analysis for resistance to other first-line and second-line drugs. Simultaneously, CDC does growth-based phenotypic drug susceptibility testing (DST) by the indirect agar proportion method for a panel of first-line and second-line antituberculosis drugs. We reviewed discordance between molecular and phenotypic DST for INH and RMP for 285 isolates submitted as MTBC to CDC from September 2009 to February 2011. We compared CDC's results with those from the submitting public health laboratories (PHL). Concordances between molecular and phenotypic testing at CDC were 97.4% for RMP and 92.5% for INH resistance. Concordances between CDC's molecular testing and PHL DST results were 93.9% for RMP and 90.0% for INH. Overall concordance between CDC molecular and PHL DST results was 91.7% for RMP and INH collectively. Discordance was primarily attributable to the absence of known INH resistance mutations in isolates found to be INH resistant by DST and detection of mutations associated with low-level RMP resistance in isolates that were RMP susceptible by phenotypic DST. Both molecular and phenotypic test results should be considered for the diagnosis of MDR TB.

New insights into fluoroquinolone resistance in Mycobacterium tuberculosis: functional genetic analysis of gyrA and gyrB mutations.

Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis by use of real-time PCR and high-resolution melt analysis.

The current study describes the development of a unique real-time PCR assay for the detection of mutations conferring drug resistance in Mycobacterium tuberculosis. The rifampicin resistance determinant region (RRDR) of rpoB and specific regions of katG and the inhA promoter were targeted for the detection of rifampin (RIF) and isoniazid (INH) resistance, respectively. Additionally, this assay was multiplexed to discriminate Mycobacterium tuberculosis complex (MTC) strains from nontuberculous Mycobacteria (NTM) strains by targeting the IS6110 insertion element. High-resolution melting (HRM) analysis following real-time PCR was used to identify M. tuberculosis strains containing mutations at the targeted loci, and locked nucleic acid (LNA) probes were used to enhance the detection of strains containing specific single-nucleotide polymorphism (SNP) transversion mutations. This method was used to screen 252 M. tuberculosis clinical isolates, including 154 RIF-resistant strains and 174 INH-resistant strains based on the agar proportion method of drug susceptibility testing (DST). Of the 154 RIF-resistant strains, 148 were also resistant to INH and therefore classified as multidrug resistant (MDR). The assay demonstrated sensitivity and specificity of 91% and 98%, respectively, for the detection of RIF resistance and 87% and 100% for the detection of INH resistance. Overall, this assay showed a sensitivity of 85% and a specificity of 98% for the detection of MDR strains. This method provides a rapid, robust, and inexpensive way to detect the dominant mutations known to confer MDR in M. tuberculosis strains and offers several advantages over current molecular and culture-based techniques.

Evaluation of the TB-Biochip oligonucleotide microarray system for rapid detection of rifampin resistance in Mycobacterium tuberculosis.

The TB-Biochip oligonucleotide microarray system is a rapid system to detect mutations associated with rifampin (RIF) resistance in mycobacteria. After optimizing the system with 29 laboratory-generated rifampin-resistant mutants of Mycobacterium tuberculosis, we evaluated the performance of this test using 75 clinical isolates of Mycobacterium tuberculosis. With this small sample set, the TB-Biochip system displayed a sensitivity of 80% and a specificity of 100% relative to conventional drug susceptibility testing results for RIF resistance. For these samples (approximately 50% tested positive), the positive predictive value was 100% and the negative predictive value was 85%. Four of the seven observed discrepancies were attributed to rare and new mutations not represented in the microarray, while three of the strains with discrepant results did not carry mutations in the RIF resistance-determining region. The results of this study confirm the utility of the system for rapid detection of RIF resistance and suggest approaches to increasing its sensitivity.

ethA, inhA, and katG loci of ethionamide-resistant clinical Mycobacterium tuberculosis isolates.

Ethionamide (ETH) is a structural analog of the antituberculosis drug isoniazid (INH). Both of these drugs target InhA, an enzyme involved in mycolic acid biosynthesis. INH requires catalase-peroxidase (KatG) activation, and mutations in katG are a major INH resistance mechanism. Recently an enzyme (EthA) capable of activating ETH has been identified. We sequenced the entire ethA structural gene of 41 ETH-resistant Mycobacterium tuberculosis isolates. We also sequenced two regions of inhA and all or part of katG. The MICs of ETH and INH were determined in order to associate the mutations identified with a resistance phenotype. Fifteen isolates were found to possess ethA mutations, for all of which the ETH MICs were > or =50 microg/ml. The ethA mutations were all different, previously unreported, and distributed throughout the gene. In eight of the isolates, a missense mutation in the inhA structural gene occurred. The ETH MICs for seven of the InhA mutants were > or =100 microg/ml, and these isolates were also resistant to > or =8 microg of INH per ml. Only a single point mutation in the inhA promoter was identified in 14 isolates. A katG mutation occurred in 15 isolates, for which the INH MICs for all but 1 were > or =32 microg/ml. As expected, we found no association between katG mutation and the level of ETH resistance. Mutations within the ethA and inhA structural genes were associated with relatively high levels of ETH resistance. Approximately 76% of isolates resistant to > or =50 microg of ETH per ml had such mutations.

Incidence of gastroduodenal ulcers associated with valdecoxib compared with that of ibuprofen and diclofenac in patients with osteoarthritis.

OBJECTIVE: To determine whether valdecoxib, at chronic arthritis doses, has the characteristics of a cyclo-oxygenase 2 (COX-2) specific inhibitor, as measured by a reduced incidence of upper-gastrointestinal ulceration compared with conventional nonsteroidal anti-inflammatory drugs (NSAIDs). METHODS: This double-blind, multicentre, placebo-controlled, parallel-group study compared the incidence of gastroduodenal ulcers associated with valdecoxib 10 mg daily (q.d.) and 20 mg q.d. with that of ibuprofen 800 mg three times daily (t.i.d.) or diclofenac 75 mg twice daily (b.i.d.) when administered over a 12-week period. The incidence of gastroduodenal ulcers was assessed by upper-gastrointestinal endoscopy, performed at baseline and again at the end of week 12 (or at early study termination). Efficacy assessments were performed at baseline and at weeks 2, 6 and 12 using Patient's and Physician's Global Assessments of Arthritis. RESULTS: A total of 1052 osteoarthritis patients were enrolled into the trial. The incidence of gastroduodenal ulcers over 12 weeks was 5% in patients receiving valdecoxib 10 mg q.d., 4% in patients receiving valdecoxib 20 mg q.d., 7% in patients receiving placebo, 16% in patients receiving ibuprofen 800 mg t.i.d. (P <0.05 v. placebo), and 17% in patients receiving diclofenac 75 mg b.i.d. (P <0.05 v. placebo). The incidence of gastroduodenal ulcers at week 12 seen in the ibuprofen 800 mg t.i.d. and diclofenac 75 mg b.i.d. groups was significantly higher than that in the valdecoxib 10 mg q.d. and valdecoxib 20 mg q.d. groups (P <0.05). The incidence rates of gastroduodenal ulcers were not significantly different between the valdecoxib treatment groups or between valdecoxib- and placebo-treated patients. Efficacy responses to valdecoxib 10 mg and 20 mg q.d. were significantly greater than placebo and comparable with both ibuprofen 800 mg t.i.d. and diclofenac 75 mg b.i.d. CONCLUSIONS: The results of the study demonstrate that valdecoxib has an upper-gastrointestinal safety profile typical of a COX-2 specific inhibitor. Overall, the data indicate that administration of valdecoxib offers similar efficacy for the treatment of osteoarthritis but improved upper-gastrointestinal safety compared with the conventional NSAIDs, ibuprofen and diclofenac, based on the significantly lower incidence of gastroduodenal ulcers detected by endoscopy.

Characterization of Mycobacterium tuberculosis complex isolates from the cerebrospinal fluid of meningitis patients at six fever hospitals in Egypt.

Mycobacterium tuberculosis complex isolates from cerebrospinal fluid of 67 meningitis patients were obtained from six fever hospitals in Egypt. One M. bovis and 66 M. tuberculosis isolates were identified by PCR-restriction fragment length polymorphism (RFLP) analysis of oxyR. Among the M. tuberculosis isolates, 53 unique strain types (with 3 to 16 copies of IS6110) were found by RFLP analyses. Nine clusters (eight with two isolates each and one with six isolates) were also found. Thirty-six spoligotypes, including at least 10 that have been previously reported from other countries, were also observed. Forty-one (62.1%) of the isolates were in spoligotype clusters, and 22 (33%) of the isolates were in RFLP clusters. Fifty-one of the isolates were susceptible in vitro to all of the antituberculosis drugs tested, 11 were monoresistant to capreomycin, rifampin, isoniazid (INH), pyrazinamide, or streptomycin (STR), 4 were resistant to STR and INH, and 1 was resistant to STR, INH, and ethambutol.