Prospective evaluation of BDProbeTec strand displacement amplification (SDA) system for diagnosis of tuberculosis in non-respiratory and respiratory samples.

Nucleic acid amplification techniques (NAATs) have been demonstrated to make significant improvements in the diagnosis of tuberculosis (TB), particularly in the time to diagnosis and the diagnosis of smear-negative TB. The BD ProbeTec strand displacement amplification (SDA) system for the diagnosis of pulmonary and non-pulmonary tuberculosis was evaluated. A total of 689 samples were analysed from patients with clinically suspected TB. Compared with culture, the sensitivity and specificity for pulmonary samples were 98 and 89 %, and against final clinical diagnosis 93 and 92 %, respectively. This assay has undergone limited evaluation for non-respiratory samples and so 331 non-respiratory samples were tested, identifying those specimens that were likely to yield a useful result. These were CSF (n = 104), fine needle aspirates (n = 64) and pus (n = 41). Pleural fluid (n = 47) was identified as a poor specimen. A concern in using the SDA assay was that low-positive samples were difficult to interpret; 7.8 % of specimens fell into this category. Indeed, 64 % of the discrepant results, when compared to final clinical diagnosis, could be assigned as low-positive samples. Specimen type did not predict likelihood of a sample being in the low-positive zone. Although the manufacturers do not describe the concept of a low-positive zone, we have found that it aids clinical diagnosis

Aetiological role of viral and bacterial infections in acute adult lower respiratory tract infection (LRTI) in primary care.

BACKGROUND: Lower respiratory tract infections (LRTI) are a common reason for consulting general practitioners (GPs). In most cases the aetiology is unknown, yet most result in an antibiotic prescription. The aetiology of LRTI was investigated in a prospective controlled study. METHODS: Eighty adults presenting to GPs with acute LRTI were recruited together with 49 controls over 12 months. Throat swabs, nasal aspirates (patients and controls), and sputum (patients) were obtained and polymerase chain reaction (PCR) and reverse transcriptase polymerase chain reaction (RT-PCR) assays were used to detect Streptococcus pneumoniae, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionella pneumophila, influenza viruses (AH1, AH3 and B), parainfluenza viruses 1-3, coronaviruses, respiratory syncytial virus, adenoviruses, rhinoviruses, and enteroviruses. Standard sputum bacteriology was also performed. Outcome was recorded at a follow up visit. RESULTS: Potential pathogens were identified in 55 patients with LRTI (69%) and seven controls (14%; p<0.0001). The identification rate was 63% (viruses) and 26% (bacteria) for patients and 12% (p<0.0001) and 6% (p = 0.013), respectively, for controls. The most common organisms identified in the patients were rhinoviruses (33%), influenza viruses (24%), and Streptococcus pneumoniae (19%) compared with 2% (p<0.001), 6% (p = 0.013), and 4% (p = 0.034), respectively, in controls. Multiple pathogens were identified in 18 of the 80 LRTI patients (22.5%) and in two of the 49 controls (4%; p = 0.011). Atypical organisms were rarely identified. Cases with bacterial aetiology were clinically indistinguishable from those with viral aetiology. CONCLUSION: Patients presenting to GPs with acute adult LRTI predominantly have a viral illness which is most commonly caused by rhinoviruses and influenza viruses

Identification of 2-Aminothiazole-4-Carboxylate Derivatives Active against Mycobacterium tuberculosis H37Rv and the β-Ketoacyl-ACP Synthase mtFabH

Background Tuberculosis (TB) is a disease which kills two million people every year and infects approximately over one-third of the world's population. The difficulty in managing tuberculosis is the prolonged treatment duration, the emergence of drug resistance and co-infection with HIV/AIDS. Tuberculosis control requires new drugs that act at novel drug targets to help combat resistant forms of Mycobacterium tuberculosis and reduce treatment duration. Methodology/Principal Findings Our approach was to modify the naturally occurring and synthetically challenging antibiotic thiolactomycin (TLM) to the more tractable 2-aminothiazole-4-carboxylate scaffold to generate compounds that mimic TLM's novel mode of action. We report here the identification of a series of compounds possessing excellent activity against M. tuberculosis H37Rv and, dissociatively, against the β-ketoacyl synthase enzyme mtFabH which is targeted by TLM. Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate was found to inhibit M. tuberculosis H37Rv with an MIC of 0.06 µg/ml (240 nM), but showed no activity against mtFabH, whereas methyl 2-(2-bromoacetamido)-5-(3-chlorophenyl)t​hiazole-4-carboxylateinhibited mtFabH with an IC50 of 0.95±0.05 µg/ml (2.43±0.13 µM) but was not active against the whole cell organism. Conclusions/Significance These findings clearly identify the 2-aminothiazole-4-carboxylate scaffold as a promising new template towards the discovery of a new class of anti-tubercular agents

A comparison of liquid and solid culture for determining relapse and durable cure in phase III TB trials for new regimens

BACKGROUND: Tuberculosis kills more people than any other infectious disease, and new regimens are essential. The primary endpoint for confirmatory phase III trials for new regimens is a composite outcome that includes bacteriological treatment failure and relapse. Culture methodology is critical to the primary trial outcome. Patients in clinical trials can have positive cultures after treatment ends that may not necessarily indicate relapse, which was ascribed previously to laboratory cross-contamination or breakdown of old lesions. Löwenstein-Jensen (LJ) medium was the previous standard in clinical trials, but almost all current and future trials will use the Mycobacteria Growth Indicator Tube (MGIT) system due to its simplicity and consistency of use, which will affect phase III trial results. LJ was used for the definition of the primary endpoint in the REMoxTB trial, but every culture was also inoculated in parallel into the MGIT system. The data from this trial, therefore, provide a unique opportunity to investigate and compare the incidence of false 'isolated positives' in liquid and solid media and their potential impact on the primary efficacy results. METHODS: All post-treatment positive cultures were reviewed in the REMoxTB clinical trial. Logistic regression models were used to model the incidence of isolated positive cultures on MGIT and LJ. RESULTS: A total of 12,209 sputum samples were available from 1652 patients; cultures were more often positive on MGIT than LJ. In 1322 patients with a favourable trial outcome, 126 (9.5%) had cultures that were positive in MGIT compared to 34 (2.6%) patients with positive cultures on LJ. Among patients with a favourable outcome, the incidence of isolated positives on MGIT differed by study laboratory (p < 0.0001) with 21.9% of these coming from one laboratory investigating only 4.9% of patients. No other baseline factors predicted isolated positives on MGIT after adjusting for laboratory. There was evidence of clustering of isolated positive cultures in some patients even after adjusting for laboratory, p < 0.0001. The incidence of isolated positives on MGIT did not differ by treatment arm (p = 0.845, unadjusted). Compared to negative MGIT cultures, positive MGIT cultures were more likely to be associated with higher grade TB symptoms reported within 7 days either side of sputum collection in patients with an unfavourable primary outcome (p < 0.0001) but not in patients with a favourable outcome (p = 0.481). CONCLUSIONS: Laboratory cross-contamination was a likely cause of isolated positive MGIT cultures which were clustered in some laboratories. Certain patients had repeated positive MGIT cultures that did not meet the definition of a relapse. This pattern was too common to be explained by cross-contamination only, suggesting that host factors were also responsible. We conclude that MGIT can replace LJ in phase III TB trials, but there are implications for the definition of the primary outcome and patient management in trials in such settings. Most importantly, the methodologies differ in the incidence of isolated positives and in their capacity for capturing non-tuberculosis mycobacteria. It emphasises the importance of effective medical monitoring after treatment ends and consideration of clinical signs and symptoms for determining treatment failure and relapse

Effects of study design and allocation on participant behaviour-ESDA: study protocol for a randomized controlled trial

Background: What study participants think about the nature of a study has been hypothesised to affect subsequent behaviour and to potentially bias study findings. In this trial we examine the impact of awareness of study design and allocation on participant drinking behaviour. Methods/Design: A three-arm parallel group randomised controlled trial design will be used. All recruitment, screening, randomisation, and follow-up will be conducted on-line among university students. Participants who indicate a hazardous level of alcohol consumption will be randomly assigned to one of three groups. Group A will be informed their drinking will be assessed at baseline and again in one month (as in a cohort study design). Group B will be told the study is an intervention trial and they are in the control group. Group C will be told the study is an intervention trial and they are in the intervention group. All will receive exactly the same brief educational material to read. After one month, alcohol intake for the past 4 weeks will be assessed. Discussion: The experimental manipulations address subtle and previously unexplored ways in which participant behaviour may be unwittingly influenced by standard practice in trials. Given the necessity of relying on self-reported outcome, it will not be possible to distinguish true behaviour change from reporting artefact. This does not matter in the present study, as any effects of awareness of study design or allocation involve bias that is not well understood. There has been little research on awareness effects, and our outcomes will provide an indication of the possible value of further studies of this type and inform hypothesis generation

Myosin-I nomenclature.

We suggest that the vertebrate myosin-I field adopt a common nomenclature system based on the names adopted by the Human Genome Organization (HUGO). At present, the myosin-I nomenclature is very confusing; not only are several systems in use, but several different genes have been given the same name. Despite their faults, we believe that the names adopted by the HUGO nomenclature group for genome annotation are the best compromise, and we recommend universal adoption

Spot sputum samples are at least as good as early morning samples for identifying Mycobacterium tuberculosis

Background The use of early morning sputum samples (EMS) to diagnose tuberculosis (TB) can result in treatment delay given the need for the patient to return to the clinic with the EMS, increasing the chance of patients being lost during their diagnostic workup. However, there is little evidence to support the superiority of EMS over spot sputum samples. In this new analysis of the REMoxTB study, we compare the diagnostic accuracy of EMS with spot samples for identifying Mycobacterium tuberculosis pre- and post-treatment. Methods Patients who were smear positive at screening were enrolled into the study. Paired sputum samples (one EMS and one spot) were collected at each trial visit pre- and post-treatment. Microscopy and culture on solid LJ and liquid MGIT media were performed on all samples; those missing corresponding paired results were excluded from the analyses. Results Data from 1115 pre- and 2995 post-treatment paired samples from 1931 patients enrolled in the REMoxTB study were analysed. Patients were recruited from South Africa (47%), East Africa (21%), India (20%), Asia (11%), and North America (1%); 70% were male, median age 31 years (IQR 24–41), 139 (7%) co-infected with HIV with a median CD4 cell count of 399 cells/μL (IQR 318–535). Pre-treatment spot samples had a higher yield of positive Ziehl–Neelsen smears (98% vs. 97%, P = 0.02) and LJ cultures (87% vs. 82%, P = 0.006) than EMS, but there was no difference for positivity by MGIT (93% vs. 95%, P = 0.18). Contaminated and false-positive MGIT were found more often with EMS rather than spot samples. Surprisingly, pre-treatment EMS had a higher smear grading and shorter time-to-positivity, by 1 day, than spot samples in MGIT culture (4.5 vs. 5.5 days, P < 0.001). There were no differences in time to positivity in pre-treatment LJ culture, or in post-treatment MGIT or LJ cultures. Comparing EMS and spot samples in those with unfavourable outcomes, there were no differences in smear or culture results, and positive results were not detected earlier in Kaplan–Meier analyses in either EMS or spot samples. Conclusions Our data do not support the hypothesis that EMS samples are superior to spot sputum samples in a clinical trial of patients with smear positive pulmonary TB. Observed small differences in mycobacterial burden are of uncertain significance and EMS samples do not detect post-treatment positives any sooner than spot samples

Four-month moxifloxacin-based regimens for drug-sensitive tuberculosis

BACKGROUND: Early-phase and preclinical studies suggest that moxifloxacin-containing regimens could allow for effective 4-month treatment of uncomplicated, smear-positive pulmonary tuberculosis. METHODS: We conducted a randomized, double-blind, placebo-controlled, phase 3 trial to test the noninferiority of two moxifloxacin-containing regimens as compared with a control regimen. One group of patients received isoniazid, rifampin, pyrazinamide, and ethambutol for 8 weeks, followed by 18 weeks of isoniazid and rifampin (control group). In the second group, we replaced ethambutol with moxifloxacin for 17 weeks, followed by 9 weeks of placebo (isoniazid group), and in the third group, we replaced isoniazid with moxifloxacin for 17 weeks, followed by 9 weeks of placebo (ethambutol group). The primary end point was treatment failure or relapse within 18 months after randomization. RESULTS: Of the 1931 patients who underwent randomization, in the per-protocol analysis, a favorable outcome was reported in fewer patients in the isoniazid group (85%) and the ethambutol group (80%) than in the control group (92%), for a difference favoring the control group of 6.1 percentage points (97.5% confidence interval [CI], 1.7 to 10.5) versus the isoniazid group and 11.4 percentage points (97.5% CI, 6.7 to 16.1) versus the ethambutol group. Results were consistent in the modified intention-to-treat analysis and all sensitivity analyses. The hazard ratios for the time to culture negativity in both solid and liquid mediums for the isoniazid and ethambutol groups, as compared with the control group, ranged from 1.17 to 1.25, indicating a shorter duration, with the lower bounds of the 95% confidence intervals exceeding 1.00 in all cases. There was no significant difference in the incidence of grade 3 or 4 adverse events, with events reported in 127 patients (19%) in the isoniazid group, 111 (17%) in the ethambutol group, and 123 (19%) in the control group. CONCLUSIONS: The two moxifloxacin-containing regimens produced a more rapid initial decline in bacterial load, as compared with the control group. However, noninferiority for these regimens was not shown, which indicates that shortening treatment to 4 months was not effective in this setting. (Funded by the Global Alliance for TB Drug Development and others; REMoxTB ClinicalTrials.gov number, NCT00864383.)