The Innate Immune Response in the Marmoset during the Acute Pneumonic Disease Caused by Burkholderia pseudomallei.

Burkholderia pseudomallei is the causative agent of melioidosis, a severe human infection that is difficult to treat with antibiotics and for which there is no effective vaccine. Development of novel treatments rely upon appropriately characterized animal models. The common marmoset (Callithrix jacchus) has been established at Defense Science and Technology laboratories (DSTL) as a model of melioidosis. Further analysis was performed on samples generated in these studies to provide a description of the innate immune response. Many of the immunological features described, (migration/activation of neutrophils and macrophages, activation of T cells, elevation of key cytokines IFNγ, TNF-α, IL-6, and IL-1ß) have been observed in acute melioidosis human cases and correlated with prognosis. Expression of the MHCII marker (HLA-DR) on neutrophils showed potential as a diagnostic with 80% accuracy when comparing pre- and postchallenge levels in paired blood samples. Discriminant analysis of cell surface, activation markers on neutrophils combined with levels of key cytokines, differentiated between disease states from single blood samples with 78% accuracy. These key markers have utility as a prototype postexposure, presymptomatic diagnostic. Ultimately, these data further validate the use of the marmoset as a suitable model for determining efficacy of medical countermeasures against B. pseudomallei.

A multi-country study using MALDI-TOF mass spectrometry for rapid identification of Burkholderia pseudomallei.

BACKGROUND: Burkholderia pseudomallei is the bacterial causative agent of melioidosis, a difficult disease to diagnose clinically with high mortality if not appropriately treated. Definitive diagnosis requires isolation and identification of the organism. With the increased adoption of MALDI-TOF MS for the identification of bacteria, we established a method for rapid identification of B. pseudomallei using the Vitek MS, a system that does not currently have B. pseudomallei in its in-vitro diagnostic database. RESULTS: A routine direct spotting method was employed to create spectra and SuperSpectra. An initial B. pseudomallei SuperSpectrum was created at Shoklo Malaria Research Unit (SMRU) from 17 reference isolates (46 spectra). When tested, this initial SMRU SuperSpectrum was able to identify 98.2 % (54/55) of Asian isolates, but just 46.7 % (35/75) of Australian isolates. Using spectra (430) from different reference and clinical isolates, two additional SMRU SuperSpectra were created. Using the combination of all SMRU SuperSpectra with seven existing SuperSpectra from Townsville, Australia 119 (100 %) Asian isolates and 31 (100 %) Australian isolates were correctly identified. In addition, no misidentifications were obtained when using these 11 SuperSpectra when tested with 34 isolates of other bacteria including the closely related species Burkholderia thailandensis and Burkholderia cepacia. CONCLUSIONS: This study has established a method for identification of B. pseudomallei using Vitek MS, and highlights the impact of geographical differences between strains for identification using this technique.

Triggers of breathlessness in inducible laryngeal obstruction and asthma.

BACKGROUND: Inducible laryngeal obstruction (ILO) is often misdiagnosed as, or may coexist with, asthma. Identifying differences in triggering factors may assist clinicians to differentiate between the two conditions and could give mechanistic insights. OBJECTIVE: To identify and compare patient-reported triggers in ILO and asthma. METHODS: This was a two-part study. Initially, we conducted a retrospective case note review of the triggers of ILO from endoscopically confirmed ILO patients to generate a Breathlessness Triggers Survey (BrTS). Triggers were categorized as scents, environmental factors, temperature, emotions, mechanical factors and daily activities. Secondly, ILO and/or asthma patients completed the BrTS prospectively, rating the likelihood of each item triggering their symptoms using a five-point Likert scale (strongly disagree to strongly agree). Chi-square testing was performed to compare responses by cohort. RESULTS: Data from 202 patients with ILO [73% female, mean (SD) age 53(16) years] were included in the case note review. For the prospective study, 38 patients with ILO only [63% females, age 57(16) years], 39 patients with asthma only [(56% female, age 53(13) years] and 12 patients with both ILO and asthma [83% female, mean age, 57 (14) years)] completed the BrTS. The triggers identified in the case note review were confirmed in the independent sample of patients with ILO and/or asthma and identified several difference in prevalence of the triggers between disease types. Mechanical factors (talking [P < .001], shouting [P = .007] and swallowing [P = .002]) were more common in the ILO cohort compared to patients with asthma. Environmental factors (pollen/flowers [P = .005] and damp air [P = .012]) were more common in asthma. There were no differences between groups in frequency of reporting scents as triggers (except for vinegar, more common in ILO, P = .019), temperature, emotions or daily activities. CONCLUSION: There were notable differences between patient-reported triggers of ILO and asthma, which may support clinician differential diagnosis.

Presence and Persistence of Zika Virus RNA in Semen, United Kingdom, 2016.

Zika virus RNA has been detected in semen collected several months after onset of symptoms of infection. Given the potential for sexual transmission of Zika virus and for serious fetal abnormalities resulting from infection during pregnancy, information regarding the persistence of Zika virus in semen is critical for advancing our understanding of potential risks. We tested serial semen samples from symptomatic male patients in the United Kingdom who had a diagnosis of imported Zika virus infection. Among the initial semen samples from 23 patients, Zika virus RNA was detected at high levels in 13 (56.5%) and was not detected in 9 (39.1%); detection was indeterminate in 1 sample (4.4%). After symptomatic infection, a substantial proportion of men have detectable Zika virus RNA at high copy numbers in semen during early convalescence, suggesting high risk for sexual transmission. Viral RNA clearance times are not consistent and can be prolonged.

Perspectives of patients and healthcare professionals on mHealth for asthma self-management.

Mobile healthcare (mHealth) has the potential to revolutionise the self-management of long-term medical conditions such as asthma. A user-centred design is integral if mHealth is to be embraced by patients and healthcare professionals.The aim of this study was to determine the perspectives of individuals with asthma and healthcare professionals on the use of mHealth for asthma self-management.We used a sequential exploratory mixed methods design; focus groups informed the development of questionnaires, which were disseminated to individuals with asthma and healthcare professionals.Focus group participants (18 asthma patients and five healthcare professionals) identified 12 potential uses of mHealth. Questionnaire results showed that individuals with asthma (n=186) most frequently requested an mHealth system to monitor asthma over time (72%) and to collect data to present to healthcare teams (70%). In contrast, healthcare professionals (n=63) most frequently selected a system alerting patients to deteriorating asthma control (86%) and advising them when to seek medical attention (87%). Individuals with asthma were less likely than healthcare professionals (p<0.001) to believe that assessing medication adherence and inhaler technique could improve asthma control.Our data provide strong support for mHealth for asthma self-management, but highlight fundamental differences between the perspectives of patients and healthcare professionals.

Performance of the GeneXpert Ebola Assay for Diagnosis of Ebola Virus Disease in Sierra Leone: A Field Evaluation Study.

BACKGROUND: Throughout the Ebola virus disease (EVD) epidemic in West Africa, field laboratory testing for EVD has relied on complex, multi-step real-time reverse transcription PCR (RT-PCR) assays; an accurate sample-to-answer RT-PCR test would reduce time to results and potentially increase access to testing. We evaluated the performance of the Cepheid GeneXpert Ebola assay on clinical venipuncture whole blood (WB) and buccal swab (BS) specimens submitted to a field biocontainment laboratory in Sierra Leone for routine EVD testing by RT-PCR ("Trombley assay"). METHODS AND FINDINGS: This study was conducted in the Public Health England EVD diagnostic laboratory in Port Loko, Sierra Leone, using residual diagnostic specimens remaining after clinical testing. EDTA-WB specimens (n = 218) were collected from suspected or confirmed EVD patients between April 1 and July 20, 2015. BS specimens (n = 71) were collected as part of a national postmortem screening program between March 7 and July 20, 2015. EDTA-WB and BS specimens were tested with Xpert (targets: glycoprotein [GP] and nucleoprotein [NP] genes) and Trombley (target: NP gene) assays in parallel. All WB specimens were fresh; 84/218 were tested in duplicate on Xpert to compare WB sampling methods (pipette versus swab); 43/71 BS specimens had been previously frozen. In all, 7/218 (3.2%) WB and 7/71 (9.9%) BS samples had Xpert results that were reported as "invalid" or "error" and were excluded, leaving 211 WB and 64 BS samples with valid Trombley and Xpert results. For WB, 22/22 Trombley-positive samples were Xpert-positive (sensitivity 100%, 95% CI 84.6%-100%), and 181/189 Trombley-negative samples were Xpert-negative (specificity 95.8%, 95% confidence interval (CI) 91.8%-98.2%). Seven of the eight Trombley-negative, Xpert-positive (Xpert cycle threshold [Ct] range 37.7-43.4) WB samples were confirmed to be follow-up submissions from previously Trombley-positive EVD patients, suggesting a revised Xpert specificity of 99.5% (95% CI 97.0%-100%). For Xpert-positive WB samples (n = 22), Xpert NP Ct values were consistently lower than GP Ct values (mean difference -4.06, 95% limits of agreement -6.09, -2.03); Trombley (NP) Ct values closely matched Xpert NP Ct values (mean difference -0.04, 95% limits of agreement -2.93, 2.84). Xpert results (positive/negative) for WB sampled by pipette versus swab were concordant for 78/79 (98.7%) WB samples, with comparable Ct values for positive results. For BS specimens, 20/20 Trombley-positive samples were Xpert-positive (sensitivity 100%, 95% CI 83.2%-100%), and 44/44 Trombley-negative samples were Xpert-negative (specificity 100%, 95% CI 92.0%-100%). This study was limited to testing residual diagnostic samples, some of which had been frozen before use; it was not possible to test the performance of the Xpert Ebola assay at point of care. CONCLUSIONS: The Xpert Ebola assay had excellent performance compared to an established RT-PCR benchmark on WB and BS samples in a field laboratory setting. Future studies should evaluate feasibility and performance outside of a biocontainment laboratory setting to facilitate expanded access to testing.

Experimental Treatment of Ebola Virus Disease with TKM-130803: A Single-Arm Phase 2 Clinical Trial.

BACKGROUND: TKM-130803, a small interfering RNA lipid nanoparticle product, has been developed for the treatment of Ebola virus disease (EVD), but its efficacy and safety in humans has not been evaluated. METHODS AND FINDINGS: In this single-arm phase 2 trial, adults with laboratory-confirmed EVD received 0.3 mg/kg of TKM-130803 by intravenous infusion once daily for up to 7 d. On days when trial enrolment capacity was reached, patients were enrolled into a concurrent observational cohort. The primary outcome was survival to day 14 after admission, excluding patients who died within 48 h of admission. After 14 adults with EVD had received TKM-130803, the pre-specified futility boundary was reached, indicating a probability of survival to day 14 of ≤0.55, and enrolment was stopped. Pre-treatment geometric mean Ebola virus load in the 14 TKM-130803 recipients was 2.24 × 109 RNA copies/ml plasma (95% CI 7.52 × 108, 6.66 × 109). Two of the TKM-130803 recipients died within 48 h of admission and were therefore excluded from the primary outcome analysis. Of the remaining 12 TKM-130803 recipients, nine died and three survived. The probability that a TKM-130803 recipient who survived for 48 h will subsequently survive to day 14 was estimated to be 0.27 (95% CI 0.06, 0.58). TKM-130803 infusions were well tolerated, with 56 doses administered and only one possible infusion-related reaction observed. Three patients were enrolled in the observational cohort, of whom two died. CONCLUSIONS: Administration of TKM-130803 at a dose of 0.3 mg/kg/d by intravenous infusion to adult patients with severe EVD was not shown to improve survival when compared to historic controls. TRIAL REGISTRATION: Pan African Clinical Trials Registry PACTR201501000997429.

Evaluation of the Biofire FilmArray BioThreat-E Test (v2.5) for Rapid Identification of Ebola Virus Disease in Heat-Treated Blood Samples Obtained in Sierra Leone and the United Kingdom.

Rapid Ebola virus (EBOV) detection is crucial for appropriate patient management and care. The performance of the FilmArray BioThreat-E test (v2.5) using whole-blood samples was evaluated in Sierra Leone and the United Kingdom and was compared with results generated by a real-time Ebola Zaire PCR reference method. Samples were tested in diagnostic laboratories upon availability, included successive samples from individual patients, and were heat treated to facilitate EBOV inactivation prior to PCR. The BioThreat-E test had a sensitivity of 84% (confidence interval [CI], 64% to 95%) and a specificity of 89% (CI, 73% to 97%) in Sierra Leone (n = 60; 44 patients) and a sensitivity of 75% (CI, 19% to 99%) and a specificity of 100% (CI, 97% to 100%) in the United Kingdom (n = 108; 70 patients) compared to the reference real-time PCR. Statistical analysis (Fisher's exact test) indicated there was no significant difference between the methods at the 99% confidence level in either country. In 9 discrepant results (5 real-time PCR positives and BioThreat-E test negatives and 4 real-time PCR negatives and BioThreat-E test positives), the majority (n = 8) were obtained from samples with an observed or probable low viral load. The FilmArray BioThreat-E test (v2.5) therefore provides an attractive option for laboratories (either in austere field settings or in countries with an advanced technological infrastructure) which do not routinely offer an EBOV diagnostic capability.

Selective adaptation to "oddball" sounds by the human auditory system.

Adaptation to both common and rare sounds has been independently reported in neurophysiological studies using probabilistic stimulus paradigms in small mammals. However, the apparent sensitivity of the mammalian auditory system to the statistics of incoming sound has not yet been generalized to task-related human auditory perception. Here, we show that human listeners selectively adapt to novel sounds within scenes unfolding over minutes. Listeners' performance in an auditory discrimination task remains steady for the most common elements within the scene but, after the first minute, performance improves for distinct and rare (oddball) sound elements, at the expense of rare sounds that are relatively less distinct. Our data provide the first evidence of enhanced coding of oddball sounds in a human auditory discrimination task and suggest the existence of an adaptive mechanism that tracks the long-term statistics of sounds and deploys coding resources accordingly.

Melioidosis diagnostic workshop, 2013.

Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions.

Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer.

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.

Liposome encapsulation of ciprofloxacin improves protection against highly virulent Francisella tularensis strain Schu S4.

Liposome-encapsulated ciprofloxacin for inhalation (CFI) was investigated as a putative postexposure therapeutic for two strains of Francisella tularensis. The efficacies of oral ciprofloxacin and intranasally instilled CFI could not be distinguished in a mouse model of infection with the F. tularensis live vaccine strain (LVS), where a single dose of either formulation offered full protection against a lethal challenge. However, mouse studies with the more virulent Schu S4 strain of F. tularensis demonstrated that a higher level of protection against a lethal aerosol infection is provided by CFI than by oral ciprofloxacin. In addition, using this infection model, it was possible to discriminate the efficacy of intranasally instilled CFI from that of aerosolized CFI, with aerosolized CFI providing full protection after just a single dose. The improved efficacy of CFI compared to oral ciprofloxacin is likely due to the high sustained concentrations of ciprofloxacin in the lung. In summary, CFI may be a promising therapy, perhaps enabling the prophylactic regimen to be shortened, for use in the event of a deliberate release of F. tularensis. The prophylactic efficacy of CFI against other biological warfare (BW) threat agents also warrants investigation.

CD99 is upregulated in placenta and astrocytomas with a differential subcellular distribution according to the malignancy stage.

In the present study, we searched for genes highly expressed in placenta and that could contribute to the establishment and maintenance of a malignant phenotype in different types of tumours, and in astrocytomas in particular. We employed a strategy based on the integration of in silico data from previously generated massively parallel signature sequencing and public serial analysis of gene expression databases. Among 12 selected genes, CD99 exhibited the highest relative mRNA expression in GBM compared to non-neoplastic brain tissues. In a larger cohort of astrocytic tumours, we further demonstrated increased CD99 expression in all malignant grades, with GBMs showing the highest values. These findings were confirmed at the protein level by Western blotting and immunohistochemistry. Additionally, we demonstrated the CD99 localisation profile in astrocytic tumours. Interestingly, CD99 expression was confined to the cytoplasm or membrane in more malignant astrocytomas, in contrast to non-neoplastic brain tissue or non-infiltrative pilocytic astrocytoma, which showed no obvious staining in these structures. Comparison of three GBM cell lines revealed higher CD99 expression at the membrane and higher migratory capacity in the A172 and U87MG lines, but lower CD99 expression and no migratory ability in the T98 line. Knocking down CD99 expression by siRNA decreased significantly the migration of both cell lines. These integrated CD99 gene and protein expression results suggest that CD99 expression in astrocytomas of different malignant grades might contribute to the infiltrative ability and support the importance of CD99 as a potential target to reduce infiltrative astrocytoma capacity in migration and invasion.

Cancer/testis antigens, gametogenesis and cancer.

Cancer/testis (CT) antigens, of which more than 40 have now been identified, are encoded by genes that are normally expressed only in the human germ line, but are also expressed in various tumour types, including melanoma, and carcinomas of the bladder, lung and liver. These immunogenic proteins are being vigorously pursued as targets for therapeutic cancer vaccines. CT antigens are also being evaluated for their role in oncogenesis--recapitulation of portions of the germline gene-expression programme might contribute characteristic features to the neoplastic phenotype, including immortality, invasiveness, immune evasion, hypomethylation and metastatic capacity.

Clinical Characteristics and Impact of Inducible Laryngeal Obstruction in the UK National Registry.

BACKGROUND: Inducible laryngeal obstruction (ILO) describes inappropriate laryngeal closure during respiration, with airflow obstruction occurring at the glottic and/or supraglottic level, leading to breathlessness. OBJECTIVE: There is a paucity of data describing the demographics and impact of ILO. We aimed to report the clinical and demographic features of ILO in individuals entered prospectively in the UK national ILO registry. METHODS: Data were entered into a Web-based registry from participants with endoscopically confirmed ILO who were attending four established UK-wide specialist ILO centers between March 2017 and November 2019. All patients provided written informed consent. RESULTS: Data from 137 individuals were included. Most (87%) had inspiratory ILO and required provocation during endoscopy to induce symptoms. There was a female predominance (80%), mean age 47 years (SD, 15 years). The most common comorbidities included asthma (68%) and reflux (57%). Health care use was high: 88% had attended emergency health care with symptoms at least once in the previous 12 months and nearly half had been admitted to the hospital. A fifth had required admission to critical care owing to ILO symptoms. Patient morbidity was substantial; 64% reported impaired functional capacity (≥3 on the Medical Research Council Dyspnoea Scale) and a third stated that symptoms affected working capability. CONCLUSION: We describe the first multicenter prospective characterization of individuals with endoscopically diagnosed ILO. Analysis of our multicenter data set identified ILO as associated with a high burden of morbidity and health care use, comparable to severe asthma. These data will support the development of health care resources in the future and guide research priorities.

Telerehabilitation for patients who have been hospitalised with covid-19: a qualitative study.

PURPOSE: The aim of this qualitative study was to explore the views of participants of a group-based, supervised, telerehabilitation programme, following discharge from hospital with Covid-19. This study was part of a single-centre, fast-track (wait-list), randomised, mixed-methods, feasibility trial of telerehabilitation (Registration: Clinicaltrials.gov reference:285205). METHODS: Semi-structured interviews were conducted over a virtual teleconference platform with 10 participants who took part in a telerehabilitation programme following Covid-19 after discharge from an acute hospital. Data were transcribed verbatim and analysed using thematic analysis. RESULTS: Five themes were important from the participant perspective: telerehabilitation programme as part of the Covid-19 journey; the telerehabilitation programme design and delivery; peer aspects; the role of the instructor; and the role of technology and online delivery. CONCLUSIONS: Overall, the telerehabilitation programme was a positive experience for participants. The instructors were central to this positive view as was the group nature of the programme. The group aspect was particularly important in supporting the broader perceived wellbeing gains, such as the sense of enjoyment and reduced social isolation. Several participants would have liked to have continued with the exercises beyond the six-week intervention indicating that the programme could be a way to help people sustain a physically active lifestyle.IMPLICATIONS FOR REHABILITATIONParticipants who were recovering from Covid-19 following hospital admission perceived the telerehabilitation to be a positive experience overall.The group aspect of the telerehabilitation programme was important in supporting the broader perceived wellbeing gains such as the sense of enjoyment and reduced social isolation.Telerehabilitation programmes for Covid-19 may need to include pathways for participants to continue to engage in exercise beyond the time-limited six-week intervention to support ongoing self-management.

Frequent and specific immunity to the embryonal stem cell-associated antigen SOX2 in patients with monoclonal gammopathy.

Specific targets of cellular immunity in human premalignancy are largely unknown. Monoclonal gammopathy of undetermined significance (MGUS) represents a precursor lesion to myeloma (MM). We show that antigenic targets of spontaneous immunity in MGUS differ from MM. MGUS patients frequently mount a humoral and cellular immune response against SOX2, a gene critical for self-renewal in embryonal stem cells. Intranuclear expression of SOX2 marks the clonogenic CD138(-) compartment in MGUS. SOX2 expression is also detected in a proportion of CD138(+) cells in MM patients. However, these patients lack anti-SOX2 immunity. Cellular immunity to SOX2 inhibits the clonogenic growth of MGUS cells in vitro. Detection of anti-SOX2 T cells predicts favorable clinical outcome in patients with asymptomatic plasmaproliferative disorders. Harnessing immunity to antigens expressed by tumor progenitor cells may be critical for prevention and therapy of human cancer.

Distinct patterns of somatic alterations in a lymphoblastoid and a tumor genome derived from the same individual.

Although patterns of somatic alterations have been reported for tumor genomes, little is known on how they compare with alterations present in non-tumor genomes. A comparison of the two would be crucial to better characterize the genetic alterations driving tumorigenesis. We sequenced the genomes of a lymphoblastoid (HCC1954BL) and a breast tumor (HCC1954) cell line derived from the same patient and compared the somatic alterations present in both. The lymphoblastoid genome presents a comparable number and similar spectrum of nucleotide substitutions to that found in the tumor genome. However, a significant difference in the ratio of non-synonymous to synonymous substitutions was observed between both genomes (P = 0.031). Protein-protein interaction analysis revealed that mutations in the tumor genome preferentially affect hub-genes (P = 0.0017) and are co-selected to present synergistic functions (P < 0.0001). KEGG analysis showed that in the tumor genome most mutated genes were organized into signaling pathways related to tumorigenesis. No such organization or synergy was observed in the lymphoblastoid genome. Our results indicate that endogenous mutagens and replication errors can generate the overall number of mutations required to drive tumorigenesis and that it is the combination rather than the frequency of mutations that is crucial to complete tumorigenic transformation.