Targeting ectromelia virus and TNF/NF-κB or STAT3 signaling for effective treatment of viral pneumonia.

Viral causes of pneumonia pose constant threats to global public health, but there are no specific treatments currently available for the condition. Antivirals are ineffective when administered late after the onset of symptoms. Pneumonia is caused by an exaggerated inflammatory cytokine response to infection, but tissue necrosis and damage caused by virus also contribute to lung pathology. We hypothesized that viral pneumonia can be treated effectively if both virus and inflammation are simultaneously targeted. Combined treatment with the antiviral drug cidofovir and etanercept, which targets tumor necrosis factor (TNF), down-regulated nuclear factor kappa B-signaling and effectively reduced morbidity and mortality during respiratory ectromelia virus (ECTV) infection in mice even when treatment was initiated after onset of clinical signs. Treatment with cidofovir alone reduced viral load, but animals died from severe lung pathology. Treatment with etanercept had no effect on viral load but diminished levels of inflammatory cytokines and chemokines including TNF, IL-6, IL-1ß, IL-12p40, TGF-ß, and CCL5 and dampened activation of the STAT3 cytokine-signaling pathway, which transduces signals from multiple cytokines implicated in lung pathology. Consequently, combined treatment with a STAT3 inhibitor and cidofovir was effective in improving clinical disease and lung pathology in ECTV-infected mice. Thus, the simultaneous targeting of virus and a specific inflammatory cytokine or cytokine-signaling pathway is effective in the treatment of pneumonia. This approach might be applicable to pneumonia caused by emerging and re-emerging viruses, like seasonal and pandemic influenza A virus strains and severe acute respiratory syndrome coronavirus 2.

Comparative genomic analyses of multi-drug resistant Mycobacterium tuberculosis from Nepal and other geographical locations.

Nepal exhibits a tuberculosis (TB) incidence rate that is comparable to neighbouring high TB incidence countries. In addition, it records >500 cases of multi-drug resistant (MDR) TB each year. The objective of this study was to perform whole-genome bioinformatic analysis on MDR-TB isolates from Nepal (n = 19) to identify the specific mutations underlying their phenotypic resistance. In addition, we examined the dominant genotype among the Nepal MDR-TB isolates, the East-Asian Beijing sub-lineage, to determine its relatedness to a panel of 1274 genomes of international strains available from public databases. These analyses provided evidence that the XDR-TB isolates in our collection were not derived from importation of primary XDR-TB to Nepal but were more likely the result of acquisition of second-line drug resistance in Nepal. Resistance to fluoroquinolones was detected among a high proportion of the Nepal isolates. This has implications for the management of TB, including appropriate antimicrobial stewardship and susceptibility testing for fluoroquinolones and other second-line TB drugs, to minimise the development of XDR-TB among Nepal TB cases.

Temporal upregulation of host surface receptors provides a window of opportunity for bacterial adhesion and disease.

Host surface receptors provide bacteria with a foothold from which to attach, colonize and, in some cases, invade tissue and elicit human disease. In this review, we discuss several key host receptors and cognate adhesins that function in bacterial pathogenesis. In particular, we examine the elevated expression of host surface receptors such as CEACAM-1, CEACAM-6, ICAM-1 and PAFR in response to specific stimuli. We explore how upregulated receptors, in turn, expose the host to a range of bacterial infections in the respiratory tract. It is apparent that exploitation of receptor induction for bacterial adherence is not unique to one body system, but is also observed in the central nervous, gastrointestinal and urogenital systems. Prokaryotic pathogens which utilize this mechanism for their infectivity include Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. A number of approaches have been used, in both in vitro and in vivo experimental models, to inhibit bacterial attachment to temporally expressed host receptors. Some of these novel strategies may advance future targeted interventions for the prevention and treatment of bacterial disease.

Therapeutic Targeting of Inflammation and Virus Simultaneously Ameliorates Influenza Pneumonia and Protects from Morbidity and Mortality.

Influenza pneumonia is a severe complication caused by inflammation of the lungs following infection with seasonal and pandemic strains of influenza A virus (IAV), that can result in lung pathology, respiratory failure, and death. There is currently no treatment for severe disease and pneumonia caused by IAV. Antivirals are available but are only effective if treatment is initiated within 48 h of onset of symptoms. Influenza complications and mortality are often associated with high viral load and an excessive lung inflammatory cytokine response. Therefore, we simultaneously targeted the virus and inflammation. We used the antiviral oseltamivir and the anti-inflammatory drug etanercept to dampen TNF signaling after the onset of clinical signs to treat pneumonia in a mouse model of respiratory IAV infection. The combined treatment down-regulated the inflammatory cytokines TNF, IL-1ß, IL-6, and IL-12p40, and the chemokines CCL2, CCL5, and CXCL10. Consequently, combined treatment with oseltamivir and a signal transducer and activator of transcription 3 (STAT3) inhibitor effectively reduced clinical disease and lung pathology. Combined treatment using etanercept or STAT3 inhibitor and oseltamivir dampened an overlapping set of cytokines. Thus, combined therapy targeting a specific cytokine or cytokine signaling pathway and an antiviral drug provide an effective treatment strategy for ameliorating IAV pneumonia. This approach might apply to treating pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

A Taxonomic and Phylogenetic Classification of Diverse Base Editors.

Base editors mediate the targeted conversion of single nucleobases in a therapeutically relevant manner. Herein, we present a hypothetical taxonomic and phylogenetic framework for the classification of more than 200 different DNA base editors, and we categorize them based on their described properties. Following evaluation of their in situ activity windows, which were derived by cataloguing their activity in published literature, organization is done hierarchically, with specific base editor signatures being subcategorized according to their on-target activity or nonspecific, genome- or transcriptome-wide activity. Based on this categorization, we curate a phylogenetic framework, based on protein homology alignment, and describe a taxonomic structure that clusters base editor variants on their target chemistry, endonuclease component, identity of their deaminase component, and their described properties into discrete taxa. Thus, we establish a hypothetical taxonomic structure that can describe and organize current and potentially future base editing variants into clearly defined groups that are defined by their characteristics. Finally, we summarize our findings into a navigable database (ShinyApp in R) that allows users to select through our repository to nominate ideal base editor candidates as a starting point for further testing in their specific application.

Draft genome sequence of a nontypeable Haemophilus influenzae strain used in the study of human respiratory infection.

OBJECTIVES: Nontypeable Haemophilus influenzae (NTHi) is an important human respiratory bacterium that can cause a range of diseases including sinusitis, otitis media, conjunctivitis, pneumonia as well as acute exacerbations of chronic obstructive pulmonary disease (COPD). A number of studies have used NTHi clinical isolate RHH-3 as a laboratory strain for experimentation examining the effect of cigarette smoke and more recently, biomass smoke, on the susceptibility and response of cells lining the respiratory tract to infection. Therefore, definition of the genome content of RHH-3 is required to fully elucidate human-NTHi interactions associated with initial infection and subsequent development of respiratory disease. DATA DESCRIPTION: Here, we present the draft genome sequence of NTHi RHH-3 collected from the sputum of a patient at the Royal Hobart Hospital, Tasmania, Australia. The assembled genome size was 1,839,376 bp consisting of 61 contigs (> 500 bp), with a G+C content of 38.1%. This draft genome data can be accessed at DDBJ/ENA/GenBank under the accession number JADPRR000000000.

Smoking and COVID-19: What we know so far.

The ongoing COVID-19 pandemic has placed a spotlight on infectious diseases and their associations with host factors and underlying conditions. New data on the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus are entering the public domain at a rapid rate such that their distillation often lags behind. To minimise weak associations becoming perceived as established paradigms, it is imperative that methodologies and outputs from different studies are appropriately critiqued and compared. In this review, we examine recent data on a potential relationship between smoking and COVID-19. While the causal role of smoking has been firmly demonstrated in regard to lung cancer and chronic obstructive pulmonary disease, such associations have the benefit of decades' worth of multi-centre epidemiological and mechanistic data. From our analysis of the available studies to date, it appears that a relationship is emerging in regard to patients with a smoking history having a higher likelihood of developing more severe symptoms of COVID-19 disease than non-smokers. Data on whether COVID-19 has a greater incidence in smokers than non-smokers is thus far, contradictory and inconclusive. There is therefore a need for some caution to be exercised until further research has been conducted in a wider range of geographical settings with sufficient numbers of patients that have been carefully phenotyped in respect of smoking status and adequate statistical control for confounding factors.

Draft Genome Sequence of an Isolate of Nontypeable Haemophilus influenzae from an Acute Exacerbation of Chronic Obstructive Pulmonary Disease in Tasmania.

Nontypeable Haemophilus influenzae (NTHi) is an important cause of human illness, including pneumonia and acute exacerbations of chronic obstructive pulmonary disease (COPD). We report here the draft genome of an isolate of NTHi collected from the sputum of a patient presenting with COPD in Tasmania, Australia.

Whole-genome analyses reveal gene content differences between nontypeable Haemophilus influenzae isolates from chronic obstructive pulmonary disease compared to other clinical phenotypes.

Nontypeable Haemophilus influenzae (NTHi) colonizes human upper respiratory airways and plays a key role in the course and pathogenesis of acute exacerbations of chronic obstructive pulmonary disease (COPD). Currently, it is not possible to distinguish COPD isolates of NTHi from other clinical isolates of NTHi using conventional genotyping methods. Here, we analysed the core and accessory genome of 568 NTHi isolates, including 40 newly sequenced isolates, to look for genetic distinctions between NTHi isolates from COPD with respect to other illnesses, including otitis media, meningitis and pneumonia. Phylogenies based on polymorphic sites in the core-genome did not show discrimination between NTHi strains collected from different clinical phenotypes. However, pan-genome-wide association studies identified 79 unique NTHi accessory genes that were significantly associated with COPD. Furthermore, many of the COPD-related NTHi genes have known or predicted roles in virulence, transmembrane transport of metal ions and nutrients, cellular respiration and maintenance of redox homeostasis. This indicates that specific genes may be required by NTHi for its survival or virulence in the COPD lung. These results advance our understanding of the pathogenesis of NTHi infection in COPD lungs.

A step-by-step beginner's protocol for whole genome sequencing of human bacterial pathogens.

Bacterial whole genome sequencing (WGS) is becoming a widely-used technique in research, clinical diagnostic, and public health laboratories. It enables high resolution characterization of bacterial pathogens in terms of properties that include antibiotic resistance, molecular epidemiology, and virulence. The introduction of next-generation sequencing instrumentation has made WGS attainable in terms of costs. However, the lack of a beginner's protocol for WGS still represents a barrier to its adoption in some settings. Here, we present detailed step-by-step methods for obtaining WGS data from a range of different bacteria (Gram-positive, Gram-negative, and acid-fast) using the Illumina platform. Modifications have been performed with respect to DNA extraction and library normalization to maximize the output from the laboratory consumables invested. The protocol represents a simplified and reproducible method for producing high quality sequencing data. The key advantages of this protocol include: simplicity of the protocol for users with no prior genome sequencing experience and reproducibility of the protocol across a wide range of bacteria.

The role of environmental exposure to non-cigarette smoke in lung disease.

Chronic exposure to household indoor smoke and outdoor air pollution is a major contributor to global morbidity and mortality. The majority of these deaths occur in low and middle-income countries. Children, women, the elderly and people with underlying chronic conditions are most affected. In addition to reduced lung function, children exposed to biomass smoke have an increased risk of developing lower respiratory tract infections and asthma-related symptoms. In adults, chronic exposure to biomass smoke, ambient air pollution, and opportunistic exposure to fumes and dust are associated with an increased risk of developing chronic bronchitis, chronic obstructive pulmonary disease (COPD), lung cancer and respiratory infections, including tuberculosis. Here, we review the evidence of prevalence of COPD in people exposed to non-cigarette smoke. We highlight mechanisms that are likely involved in biomass-smoke exposure-related COPD and other lung diseases. Finally, we summarize the potential preventive and therapeutic strategies for management of COPD induced by non-cigarette smoke exposure.

Diagnostic Accuracy of GeneXpert MTB/RIF Assay in Comparison to Conventional Drug Susceptibility Testing Method for the Diagnosis of Multidrug-Resistant Tuberculosis.

Xpert MTB/RIF assay is regarded as a great achievement of modern medicine for the rapid diagnosis of multidrug-resistant tuberculosis (MDR-TB). The main purpose of this study was to determine the performance of Xpert MTB/RIF assay compared to conventional drug susceptibility testing (DST) method for the diagnosis of MDR-TB. A comparative cross sectional study was carried out at German-Nepal Tuberculosis Project, Kathmandu, Nepal, from April 2014 to September 2014. A total of 88 culture positive clinical samples (83 pulmonary and 5 extra-pulmonary) received during the study period were analyzed for detection of multidrug-resistant tuberculosis by both GeneXpert MTB/RIF assay and conventional DST method. McNemar chi square test was used to compare the performance of Xpert with that of DST method. A p-value of less than 0.05 was considered as statistically significant. Of total 88 culture positive samples, one was reported as invalid while 2 were found to contain nontuberculous Mycobacteria (NTM). Among remaining 85 Mycobacterium tuberculosis culture positive samples, 69 were found to be MDR-TB positive by both methods. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of GeneXpert MTB/RIF assay were found to be 98.6%, 100%, 100% and 93.8% respectively. Statistically, there was no significant difference between the diagnostic performance of Xpert and conventional DST method for detection of MDR-TB. GeneXpert MTB/RIF assay was found to be highly sensitive, specific and comparable to gold standard conventional DST method for the diagnosis of MDR-TB.

Thyroid dysfunction in metabolic syndrome patients and its relationship with components of metabolic syndrome.

BACKGROUND: A growing body of evidence suggests that metabolic syndrome is associated with endocrine disorders including thyroid dysfunction. Thyroid dysfunction in metabolic syndrome patients may further add to cardiovascular disease risk thereby increasing mortality. This study was done to assess thyroid function in metabolic syndrome patients and evaluate its relationship with the components of metabolic syndrome. METHODS: A cross sectional study was carried out among 169 metabolic syndrome patients at B P Koirala Institute of Health Sciences, Dharan, Nepal. Anthropometric measurements (height, weight, waist circumference) and blood pressure were taken. Fasting blood samples were analysed to measure glucose, triglyceride, high density lipoprotein (HDL) cholesterol and thyroid hormones (triiodothyronine, thyroxine and thyroid stimulating hormone). RESULTS: Thyroid dysfunction was seen in 31.9 % (n = 54) metabolic syndrome patients. Subclinical hypothyroidism (26.6 %) was the major thyroid dysfunction followed by overt hypothyroidism (3.5 %) and subclinical hyperthyroidism (1.7 %). Thyroid dysfunction was much common in females (39.7 %, n = 29) than males (26 %, n = 25) but not statistically significant (p = 0.068). The relative risk of having thyroid dysfunction in females was 1.525 (CI: 0.983-2.368) as compared to males. Significant differences (p = 0.001) were observed in waist circumference between patients with and without thyroid dysfunction and HDL cholesterol which had significant negative correlation with thyroid stimulating hormone. CONCLUSIONS: Thyroid dysfunction, particularly subclinical hypothyroidism is common among metabolic syndrome patients, and is associated with some components of metabolic syndrome (waist circumference and HDL cholesterol).

Association of high-sensitivity C-reactive protein and uric acid with the metabolic syndrome components.

Metabolic syndrome (MetS) has been found to be associated with inflammatory molecules. This study was conducted among 125 MetS patients at B P Koirala Institute of Health Sciences, Dharan, Nepal to find an association of high-sensitivity C-reactive protein (hs-CRP) and serum uric acid with MetS components. Anthropometric measurements, blood pressure, medical history and blood samples were taken. Estimation of hs-CRP, serum uric acid, blood glucose, triglyceride and high density lipoprotein (HDL) cholesterol was done. hs-CRP had positive correlation with blood glucose (r = 0.2, p = 0.026) and negative with HDL cholesterol (r = -0.361, p < 0.001). Serum uric acid had positive correlation with waist circumference (r = 0.178, p = 0.047). Patients with elevated hs-CRP and uric acid had higher waist circumference (p = 0.03), diastolic BP (p = 0.002) and lower HDL cholesterol (p = 0.004) than others. Elevated hs-CRP and high uric acid were individually associated with higher odds for low HDL cholesterol (7.992; 1.785-35.774, p = 0.002) and hyperglycemia (2.471; 1.111-5.495, p = 0.029) respectively. Combined rise of hs-CRP and uric acid was associated with severity of MetS (p < 0.001) and higher odds for hyperglycemia (8.036; 2.178-29.647, p = 0.001) as compared to individual rise of hs-CRP or uric acid. The present study demonstrates that hs-CRP and serum uric acid are associated with MetS components, and the combined rise of hs-CRP and uric acid is associated with the increase in severity of MetS.

Cardiovascular Risk Factors in Subclinical Hypothyroidism: A Case Control Study in Nepalese Population.

Objectives. To assess cardiovascular risk factors in Nepalese population with subclinical hypothyroidism as compared to age and sex matched controls. Materials and Methods. A case control study was conducted among 200 subjects (100 subclinical hypothyroid and 100 euthyroid) at B.P. Koirala Institute of Health Sciences, Dharan, Nepal. Demographic and anthropometric variables including systolic and diastolic blood pressure (BP) were taken. Blood samples were assayed for serum free triiodothyronine (fT3), free thyroxine (fT4), thyroid stimulating hormone (TSH), total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and high sensitivity C reactive protein (hs-CRP). Results. Subclinical hypothyroid patients had significantly higher diastolic BP, total cholesterol, LDL cholesterol, and hs-CRP than controls. The odds ratio of having hypercholesterolemia (>200 mg/dL), low HDL cholesterol (<40 mg/dL), undesirable LDL-cholesterol (>100 mg/dL), high hs-CRP (>1 mg/L), and high diastolic BP (>80 mmHg) and being overweight (BMI ≥ 23 Kg/m(2)) in subclinical hypothyroidism was 2.29 (95% CI; 1.2-4.38, p = 0.011), 1.73 (95% CI; 0.82-3.62, p = 0.141), 3.04 (95% CI; 1.66-5.56, p < 0.001), 2.02 (95% CI; 1.12-3.64, p = 0.018), 3.35 (95% CI; 1.72-6.55, p < 0.001), and 0.9 (95% CI; 0.48-1.67, p = 0.753), respectively, as compared to controls. Conclusion. Subclinical hypothyroid patients are associated with higher risk for cardiovascular disease than euthyroid subjects.

Thyroid Dysfunction and Associated Risk Factors among Nepalese Diabetes Mellitus Patients.

Objectives. To assess thyroid function and associated risk factors in Nepalese diabetes mellitus patients. Methods. A cross-sectional study was carried out among 419 diabetes mellitus patients at B. P. Koirala Institute of Health Sciences, Dharan, Nepal. Information on demographic and anthropometric variables and risk factors for thyroid dysfunction was collected. Blood samples were analysed to measure thyroid hormones, blood sugar, and lipid profile. Results. Prevalence rate of thyroid dysfunction was 36.03%, with subclinical hypothyroidism (26.5%) as the most common thyroid dysfunction. Thyroid dysfunction was much common in females (42.85%) compared to males (30.04%) (p = 0.008) and in type 1 diabetes (50%) compared to type 2 diabetes mellitus (35.41%) (p = 0.218). Diabetic patients with thyroid dysfunction had higher total cholesterol, HDL cholesterol, and LDL cholesterol in comparison to patients without thyroid dysfunction. Significant risk factors for thyroid dysfunction, specifically hypothyroidism (overt and subclinical), were smoking (relative risk of 2.56 with 95% CI (1.99-3.29, p < 0.001)), family history of thyroid disease (relative risk of 2.57 with 95% CI (2.0-3.31, p < 0.001)), and female gender (relative risk of 1.44 with 95% CI (1.09-1.91, p = 0.01)). Conclusions. Thyroid dysfunction is common among Nepalese diabetic patients. Smoking, family history of thyroid disease, and female gender are significantly associated with thyroid dysfunction.