Chronic Pulmonary Infection Research Articles

Regulation of lung inflammation by adiponectin.

Adiponectin is an insulin sensitizing hormone that also plays a role in the regulation of inflammation. Although adiponectin can exert pro-inflammatory effects, more studies have reported anti-inflammatory effects, even in non-adipose tissues such as the lung. Obesity is considered an inflammatory disease, is a risk factor for lung diseases, and is associated with decreased levels of plasma adiponectin. The results of recent studies have suggested that adiponectin exerts anti-inflammatory activity in chronic obstructive pulmonary disease, asthma and invasive fungal infection. The signaling receptors of adiponectin, AdipoR1 and AdipoR2, are expressed by epithelial cells, endothelial cells, and immune cells in the lung. In this mini-review, we discuss the anti-inflammatory mechanisms of adiponectin in lung cells and tissues.

Changes of androgen and corticosterone metabolites excretion and conversion in cystic fibrosis.

Cystic fibrosis (CF) is a life-threatening inherited disease related to a mutation in the CFTR gene, that leads to serious health complications such as chronic pulmonary infections, pancreatic insufficiency, dysfunction of the sweat glands and reproductive system. For the first time, we have described the profile of corticosterone and androgen metabolites in urine, as well as the activity of enzymes involved in steroid genesis and metabolism in people with CF, using gas chromatography/mass spectrometry. A significant reduction in the excretion of most of the measured metabolites in CF was found. These differences were observed in the group of progestagen metabolites, as well as among metabolites of corticosterone and androgens. We revealed higher activities of 17β-hydroxysteroid dehydrogenase and 17,20-lyase in the Δ4 pathway compared with controls, what can promote the androgen synthesis through the backdoor androgen pathway. We have also found the increased conversion activity of 11-oxyganated steroids by 5a-reductase in backdoor pathway. Levels of the most potent and vital androgens (testosterone and dihydrotestosterone) are comparable in both groups. However, the excretion of dehydroepiandrosterone was lower in CF. Decreased cholesterol lipoprotein levels may contribute to limited intracellular cholesterol supply and reduced adrenal steroidogenesis in CF individuals. Changes in the activity of some steroidogenesis enzymes may suggest the presence of some peripheral adaptive mechanisms in CF to maintain androgen balance in the body despite the limited sufficiency of secretion by the adrenal cortex.

Spatial lipidomics reveals biased phospholipid remodeling in acute Pseudomonas lung infection

Pseudomonas aeruginosa (Pa) is a pathogen causing chronic pulmonary infections in patients with cystic fibrosis (CF). Manipulation of lipids is an important feature of Pa infection and on a tissue-level scale is poorly understood. Using a mouse model of acute Pa pulmonary infection, we explored the whole-lung phospholipid response using mass spectrometry imaging (MSI) and spatial lipidomics. Using a histology-driven analysis, we isolated airways and parenchyma from both mock- and Pa-infected lungs and used systems biology tools to identify enriched metabolic pathways from the differential phospholipid identities. Infection was associated with a set of 26 ions, with 11 unique to parenchyma and 6 unique to airways. Acyl remodeling was differentially enriched in infected parenchyma as the predominant biological function. These functions correlated with markers of polymorphonuclear (PMN) cell influx, a defining feature of the lung response to Pa infection, implicating enzymes active in phospholipid remodeling.

Chronic pulmonary bacterial infection facilitates breast cancer lung metastasis by recruiting tumor-promoting MHCIIhi neutrophils

Breast cancer can metastasize to various organs, including the lungs. The immune microenvironment of the organs to be metastasized plays a crucial role in the metastasis of breast cancer. Infection with pathogens such as viruses and bacteria can alter the immune status of the lung. However, the effect of chronic inflammation caused by bacteria on the formation of a premetastatic niche within the lung is unclear, and the contribution of specific immune mediators to tumor metastasis also remains largely undetermined. Here, we used a mouse model revealing that chronic pulmonary bacterial infection augmented breast cancer lung metastasis by recruiting a distinct subtype of tumor-infiltrating MHCIIhi neutrophils into the lung, which exhibit cancer-promoting properties. Functionally, MHCIIhi neutrophils enhanced the lung metastasis of breast cancer in a cell-intrinsic manner. Furthermore, we identified CCL2 from lung tissues as an important environmental signal to recruit and maintain MHCIIhi neutrophils. Our findings clearly link bacterial-immune crosstalk to breast cancer lung metastasis and define MHCIIhi neutrophils as the principal mediator between chronic infection and tumor metastasis.

Scorpionfish BPI is highly active against multiple drug-resistant Pseudomonas aeruginosa isolates from people with cystic fibrosis.

Chronic pulmonary infection is a hallmark of cystic fibrosis (CF) and requires continuous antibiotic treatment. In this context, Pseudomonas aeruginosa (Pa) is of special concern since colonizing strains frequently acquire multiple drug resistance (MDR). Bactericidal/permeability-increasing protein (BPI) is a neutrophil-derived, endogenous protein with high bactericidal potency against Gram-negative bacteria. However, a significant range of people with CF (PwCF) produce anti-neutrophil cytoplasmic antibodies against BPI (BPI-ANCA), thereby neutralizing its bactericidal function. In accordance with literature, we describe that 51.0% of a total of 39 PwCF expressed BPI-ANCA. Importantly, an orthologous protein to human BPI (huBPI) derived from the scorpionfish Sebastes schlegelii (scoBPI) completely escaped recognition by these autoantibodies. Moreover, scoBPI exhibited high anti-inflammatory potency towards Pa LPS and was bactericidal against MDR Pa derived from PwCF at nanomolar concentrations. In conclusion, our results highlight the potential of highly active orthologous proteins of huBPI in treatment of MDR Pa infections, especially in the presence of BPI-ANCA.

Loss of allosteric regulation in α-isopropylmalate synthase identified as an antimicrobial resistance mechanism

Despite our best efforts to discover new antimicrobials, bacteria have evolved mechanisms to become resistant. Resistance to antimicrobials can be attributed to innate, inducible, and acquired mechanisms. Mycobacterium abscessus is one of the most antimicrobial resistant bacteria and is known to cause chronic pulmonary infections within the cystic fibrosis community. Previously, we identified epetraborole as an inhibitor against M. abscessus with in vitro and in vivo activities and that the efficacy of epetraborole could be improved with the combination of the non-proteinogenic amino acid norvaline. Norvaline demonstrated activity against the M. abscessus epetraborole resistant mutants thus, limiting resistance to epetraborole in wild-type populations. Here we show M. abscessus mutants with resistance to epetraborole can acquire resistance to norvaline in a leucyl-tRNA synthetase (LeuRS) editing-independent manner. After showing that the membrane hydrophobicity and efflux activity are not linked to norvaline resistance, whole-genome sequencing identified a mutation in the allosteric regulatory domain of α-isopropylmalate synthase (α-IPMS). We found that mutants with the α-IPMSA555V variant incorporated less norvaline in the proteome and produced more leucine than the parental strain. Furthermore, we found that leucine can rescue growth inhibition from norvaline challenge in the parental strain. Our results demonstrate that M. abscessus can modulate its metabolism through mutations in an allosteric regulatory site to upregulate the biosynthesis of the natural LeuRS substrate and outcompete norvaline. These findings emphasize the antimicrobial resistant nature of M. abscessus and describe a unique mechanism of substrate-inhibitor competition.

Lung Microbiota: Its Relationship to Respiratory System Diseases and Approaches for Lung-Targeted Probiotic Bacteria Delivery.

Microorganisms that make up the local microbiota (such as Lactobacillus sp. and Bifidobacterium sp.) play a crucial role in the modulation of diseases and health states by taking place not only in the gut but also in many parts of our body. There is also interference between the gut and the lung via the gut-lung axis. The relationship between respiratory diseases and lung microbiota, which become more of an issue of particular importance in recent years, shows that probiotics play an essential role in maintaining the balance of microorganisms in the respiratory tract. However, studies on probiotics' prophylactic or therapeutic application in chronic lung diseases are limited. In this review, the literature between 1977 and 2022 was surveyed. General information about human microbiota was accessed in earlier sources, and especially in the past decade, research on lung microbiota has been reached. The relationship between lung microbiota and important respiratory diseases such as bronchopulmonary dysplasia, chronic obstructive pulmonary disease, pneumonia, cystic fibrosis, allergy-asthma, influenza, lung cancer, and COVID-19 infection, was scrutinized after mentioning human microbiota, the gut-lung axis, and respiratory tract microbiota. The mechanism of action of probiotics and the formulation approaches of probiotics in terms of pharmaceutical technology were reviewed. Finally, future perspectives on lung-targeted administration of probiotic bacteria with prophylactic or therapeutic potential, or both, were presented.

Foreign Body Aspiration in Children-Retrospective Study and Management Novelties.

Foreign body aspiration (FBA) is a frequent diagnosis in children. In the absence of other lung conditions, such as asthma or chronic pulmonary infections, this manifests as a sudden onset of cough, dyspnea, and wheezing. The differential diagnosis is based on a scoring system which takes into account the clinical picture as well as the radiologic aspects. The treatment that is considered the gold-standard for FBA in children remains to be rigid fibronchoscopy, however it comes with several potentially crucial local complications such as airway edema, bleeding, and bronchospasm, along inherent issues due to general anesthesia. Material and methods: Our study is a retrospective study analyzing the medical files of the cases from our hospital over the span of 9 years. The study group consisted of 242 patients aged 0-16 years diagnosed with foreign body aspiration in the Emergency Clinical Hospital for Children "Sfânta Maria" Iași, between January 2010-January 2018. Clinical and imaging data were extracted from the patients' observation sheets. Results: In our cohort, the distribution of children with foreign body aspiration was uneven, with the highest incidence being reported in children from rural areas (70% of cases), whereas the most affected age group was 1-3 years, amounting to 79% of all cases. The main symptoms which led to emergency admittance were coughing (33%) and dyspnea (22%). The most important factors that determined the unequal distribution were socio-economic status, which relates to a lack of adequate supervision by parents, as well as the consumption of food inappropriate for their age. Conclusions: Foreign body aspiration is a major medical emergency that may be associated with dramatic clinical manifestations. Several scoring algorithms designed to establish the need for bronchoscopy have been proposed, taking into account both the clinical and radiological results. The issue with asymptomatic or mild symptomatic cases, as well as difficulties managing cases with radiolucent foreign bodies, remains a challenge.

WS07.04 Hostile takeover factors of Pseudomonas aeruginosa: contact dependent secretion systems

Objectives: Pseudomonas aeruginosa is the most common cause of chronic pulmonary infections in Cystic Fibrosis (CF) patients, dominating and damaging the lung environment. P. aeruginosa can deliver bacterial-derived toxins into competing neighbours to inhibit growth or kill via the contact-dependent inhibition (CDI) systems. However, the role of CDI systems in Pseudomonas infection in Cystic Fibrosis clinical isolates and dominance during chronic infection remains unclear. Here we bioinformatically and experimentally investigate the CDI systems encoded in P. aeruginosa strain PA14.

Rifaximin potentiates clarithromycin against Mycobacterium abscessus in vitro and in zebrafish.

Mycobacterium abscessus is a non-tuberculous mycobacterium (NTM) that causes chronic pulmonary infections. Because of its extensive innate resistance to numerous antibiotics, treatment options are limited, often resulting in poor clinical outcomes. Current treatment regimens usually involve a combination of antibiotics, with clarithromycin being the cornerstone of NTM treatments. To identify drug candidates that exhibit synergistic activity with clarithromycin against M. abscessus. We performed cell-based phenotypic screening of a compound library against M. abscessus induced to become resistant to clarithromycin. Furthermore, we evaluated the toxicity and efficacy of the top compound in a zebrafish embryo infection model. The screen revealed rifaximin as a clarithromycin potentiator. The combination of rifaximin and clarithromycin was synergistic and bactericidal in vitro and potent in the zebrafish model. The data indicate that the rifaximin/clarithromycin combination is promising to effectively treat pulmonary NTM infections.

Eradication of Burkholderia cepacia complex in cystic fibrosis patients with inhalation of amiloride and tobramycin combined with oral cotrimoxazole.

This case series suggests that successful eradication therapy of BCC in cystic fibrosis can be done with a combination of inhaled and oral medication, which in many cases may eliminate the need for intensive treatment with intravenous antibiotics https://bit.ly/40oOMIn.

Antibodies against Histoplasma capsulatum and Aspergillus fumigatus among chronic TB patients in Indonesia: a cross-sectional study.

Chronic pulmonary fungal infections may occur in patients with previous history of pulmonary tuberculosis (TB), and are often clinically misclassified as TB, especially when bacteriological confirmation for Mycobacterium tuberculosis is absent. In this study, we investigated the prevalence of antibody against Histoplasma capsulatum and Aspergillus fumigatus in patients with confirmed and clinically chronic TB. Antibodies against H. capsulatum and A. fumigatus were measured from serum samples using enzyme-linked immunosorbent assay (ELISA). The presence M. tuberculosis in sputum was confirmed using smear microscopy, GeneXpert MTB/RIF assay, or culture. Antibodies against H. capsulatum and A. fumigatus were elevated in 16.9% and 26.9% of bacteriologically confirmed chronic TB patients, and 12.1% and 18.2% in those without bacteriological confirmation, respectively. Approximately one-third of patients who had positive anti-Histoplasma antibody also had elevated levels of antibody against Aspergillus fumigatus (P<.001). Our study highlights the importance of chronic pulmonary fungal infection in post-TB patients with recurrent respiratory symptoms.

Mechanism of resistance to phagocytosis and pulmonary persistence in mucoid Pseudomonas aeruginosa.

Pseudomonas aeruginosa is known for its ability to form biofilms, which are dependent on the production of exopolysaccharides. During chronic colonization of the airway and biofilm formation, P. aeruginosa converts to a mucoid phenotype, indicating production of the exopolysaccharide alginate. The mucoid phenotype promotes resistance to phagocytic killing, but the mechanism has not been established. To better understand the mechanism of phagocytic evasion conferred by alginate production, Human (THP-1) and murine (MH-S) macrophage cell lines were used to determine the effects of alginate production on macrophage binding, signaling and phagocytosis. Phagocytosis assays using mucoid clinical isolate FRD1 and its non-mucoid algD mutant showed that alginate production inhibited opsonic and non-opsonic phagocytosis, but exogenous alginate was not protective. Alginate caused a decrease in binding to murine macrophages. Blocking antibodies to CD11b and CD14 showed that these receptors were important for phagocytosis and were blocked by alginate. Furthermore, alginate production decreased the activation of signaling pathways required for phagocytosis. Mucoid and non-mucoid bacteria induced similar levels of MIP-2 from murine macrophages. This study demonstrated for the first time that alginate on the bacterial surface inhibits receptor-ligand interactions important for phagocytosis. Our data suggest that there is a selection for alginate conversion that blocks the earliest steps in phagocytosis, leading to persistence during chronic pulmonary infections.

Current and Emerging Inhaled Antibiotics for Chronic Pulmonary Pseudomonas aeruginosa and Staphylococcus aureus Infections in Cystic Fibrosis.

Characterized by impaired mucus transport and subsequent enhanced colonization of bacteria, pulmonary infection causes major morbidity and mortality in patients with cystic fibrosis (CF). Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) are the two most common types of bacteria detected in CF lungs, which undergo multiple adaptational mechanisms such as biofilm formation resulting in chronic pulmonary infections. With the advantages of greater airway concentration and minimized systemic toxicity, inhaled antibiotics are introduced to treat chronic pulmonary infection in CF. Inhaled tobramycin, aztreonam, levofloxacin, and colistin are the four most common discussed inhaled antibiotics targeting P. aeruginosa. Additionally, inhaled liposomal amikacin and murepavadin are also in development. This review will discuss the virulence factors and adaptational mechanisms of P. aeruginosa and S. aureus in CF. The mechanism of action, efficacy and safety, current status, and indications of corresponding inhaled antibiotics will be summarized. Combination therapy and the strategies to select an optimal inhaled antibiotic protocol will also be discussed.

The effects of vitamin C on respiratory, allergic and immunological diseases: an experimental and clinical-based review.

Vitamin C is used in modern medicine supplements for treatment of various disorders associated with oxidative stress, inflammation and immune dysregulation. In this review article, experimental and clinical results regarding the effects of vitamin C on respiratory immunologic, and allergic diseases are reviewed. Various databases and appropriate keywords are used to search the effect of vitamin C on respiratory diseases until the end of May 2022. Books, theses and articles were included. These studies assessed the effects of vitamin C on respiratory disorders including asthma, chronic obstructive pulmonary disease (COPD), lung infection and lung cancer. Vitamin C showed relaxant effect on tracheal smooth muscle via various mechanisms. The preventive effects of vitamin C were mediated by antioxidant, immunomodulatory and anti-inflammatory mechanisms in the experimental animal models of different respiratory diseases. Some clinical studies also indicated the effect of vitamin C on lung cancer and lung infections. Therefore, vitamin C could be used a preventive and/or relieving therapy in respiratory diseases.

Cardiovascular, respiratory and all-cause (natural) health endpoint estimation using a spatial approach in Malaysia

In 2016, the World Health Organization (WHO) estimated that approximately 4.2 million premature deaths worldwide were attributable to exposure to particulate matter 2.5 μm (PM2.5). This study assessed the environmental burden of disease attributable to PM2.5 at the national level in Malaysia. We estimated the population-weighted exposure level (PWEL) of PM10 concentrations in Malaysia for 2000, 2008, and 2013 using aerosol optical density (AOD) data from publicly available remote sensing satellite data (MODIS Terra). The PWEL was then converted to PM2.5 using Malaysia's WHO ambient air conversion factor. We used AirQ+ 2.0 software to calculate all-cause (natural), ischemic heart disease (IHD), stroke, chronic obstructive pulmonary disease (COPD), lung cancer (LC), and acute lower respiratory infection (ALRI) excess deaths from the National Burden of Disease data for 2000, 2008 and 2013. The average PWELs for annual PM2.5 for 2000, 2008, and 2013 were 22 μg m-3, 18 μg m-3 and 24 μg m-3, respectively. Using the WHO 2005 Air Quality Guideline cut-off point of PM2.5 of 10 μg m-3, the estimated excess deaths for 2000, 2008, and 2013 from all-cause (natural) mortality were between 5893 and 9781 (95 % CI: 3347–12,791), COPD was between 164 and 957 (95 % CI: 95–1411), lung cancer was between 109 and 307 (95 % CI: 63–437), IHD was between 3 and 163 deaths, according to age groups (95 % CI: 2–394) and stroke was between 6 and 155 deaths, according to age groups (95 % CI: 3–261). An increase in estimated health endpoints was associated with increased estimated PWEL PM2.5 for 2013 compared to 2000 and 2008. Adhering the ambient PM2.5 level to the Malaysian Air Quality Standard IT-2 would reduce the national health endpoints mortality.

Nanomedicine - a boon for respiratory disease management

Respiratory diseases affect the lungs and other parts of the respiratory system. The respiratory disease affects hundreds of millions of humans, and premature death is observed in nearly four million people yearly. The major cause of the increase in this disease is the increased level of air pollution and higher tobacco usage in public places.We have used the search engines PubMed and Google Scholar for the keywords Respiratory diseases, Nanomaterials, diagnosis, Nanomedicine, and Target drug delivery; recent and relevant articles are selected for reviewing this paper.Nanomedicine is a recent field of research that deals with monitoring, repairing, theragnosis, and development of human biological systems at the sub-atomic level, where we utilize engineered nanodevices and nanostructures. The conventional therapeutic strategies designed for respiratory diseases have limited solubility and bioavailability. Moreover, the robust effect of the drugs led to adverse side effects due to their high dose requirement. The local delivery of therapeutic Nanoparticles (NPs) or drug-loaded nano vehicles to the lung is a safe technique for managing various respiratory tract-related diseases like chronic obstructive pulmonary diseases, cystic fibrosis, lung cancer, tuberculosis, asthma, and infection. To overcome the difficulties of conventional treatment with antibiotics and anti-inflammatory drugs, nano-enabled drug delivery, nanoformulations of drugs as well as drug nanoencapsulation have been used recently. In this mini-review, we will discuss the importance and application of nanomedicine for diagnosis, treatment and clinical research involved in the different types of respiratory diseases.Nanomedicine provides an alternative delivery of drugs with the help of various nanocarriers, which enhances controlled drug delivery at the pulmonary region and can be used for treating and diagnosing respiratory diseases in vivo and in vitro studies. Further experiments followed by clinical examination are warranted to prove the potential application of nanomedicine in treating respiratory disease.This mini-review will help the readers and budding scientists apply new methods for developing highly efficient drugs with low side effects and improved targeted sites of action.

The Burkholderia contaminans prevalent phenotypes as possible markers of poor clinical outcomes in chronic lung infection of children with cystic fibrosis.

Burkholderia contaminans, a species of the Burkholderia cepacia complex-prevalent in certain Latin-American and European countries-can cause chronic pulmonary infection in persons with cystic fibrosis. Our aim was to gain insights into long-term lung infections with a focus on correlating how bacterial phenotypic traits in the chronic infection impact on patients´ clinical outcome. Genotypic characteristics of 85 B. contaminans isolates recovered from 70 patients were investigated. For 16 of those patients, the clinical status and bacterial phenotypic characteristics, e. g., several virulence factors, phenotypic variants, and the antimicrobial susceptibility pattern, were evaluated. Two clones were found in the whole bacterial population: i) the multiresistant ST 872 PCR-recA-RFLP-HaeIII-K-pattern clone which carries a pathogenic island homologous to BcenGI11 of B. cenocepacia J2315, and ii) the ST 102 PCR-recA-RFLP-HaeIII-AT-pattern clone. The emergence of certain bacterial phenotypes in the chronic infection such as the nonmucoid phenotype, small colony variants, brownish pigmented colonies, and hypermutators, proved to be, together with co-infection with Pseudomonas aeruginosa, the possible markers of more challenging infections and poor prognosis. The presence of co-colonizers and the bacterial phenotypes that are especially adapted to persist in long-term respiratory tract infections have a crucial role in patients' clinical outcomes.

Role of inhaled antibiotics in the era of highly effective CFTR modulators.

Recurrent and chronic bacterial infections are common in people with cystic fibrosis (CF) and contribute to lung function decline. Antibiotics are the mainstay in the treatment of exacerbations and chronic bacterial infection in CF. Inhaled antibiotics are effective in treating chronic respiratory bacterial infections and eradicating Pseudomonas aeruginosa from the respiratory tract, with limited systemic adverse effects. In the past decade, highly effective cystic fibrosis transmembrane conductance regulator (CFTR) modulators have become a new therapy that partially corrects/opens chloride transport in patients with selected CFTR mutations, restoring mucus hydration and improving mucociliary clearance. The recent triple CFTR modulator combination is approved for ∼80-90% of the CF population and significantly reduces pulmonary exacerbations and improves respiratory symptoms and lung function. CFTR modulators have shifted the focus from symptomatic treatment to personalised/precision medicine by targeting genotype-specific CFTR defects. While these are highly effective, they do not fully normalise lung physiology, stop inflammation or resolve chronic lung damage, such as bronchiectasis. The impact of these new drugs on lung health is likely to change the future management of chronic pulmonary infections in people with CF. This article reviews the role of inhaled antibiotics in the era of CFTR modulators.

The Incidence and Risk Factors of Chronic Pulmonary Infection after Radiotherapy in Patients with Lung Cancer.

This study aimed to investigate cumulative incidence and risk factors associated with chronic pulmonary infection (CPI) development after radiotherapy for lung cancer. We retrospectively analyzed 1,872 patients with lung cancer who received radiotherapy for lung cancer from 2010-2014, had a follow-up period of ≥ 3 months after radiotherapy, and did not have CPI at the time of radiotherapy. CPI was defined as pulmonary tuberculosis, non-tuberculous mycobacterial pulmonary disease, chronic pulmonary aspergillosis, or pulmonary actinomycosis. The cumulative incidence of CPI and overall survival (OS) were estimated using the Kaplan-Meier method, and a multivariable Cox proportional hazards analysis was performed to identify risk factors associated with CPI development. The median follow-up period was 2.3 years with OS rates of 55.6% and 37.6% at 2 and 5 years, respectively. CPI developed in 59 patients at a median of 1.8 years after radiotherapy, with cumulative incidence rates of 1.1%, 3.4%, 5.0%, and 6.8% at 1, 3, 5, and 7 years, respectively. A lower body mass index, interstitial lung disease, prior pulmonary tuberculosis, larger clinical target volume, history of lung cancer surgery or radiation pneumonitis, and use of inhaled corticosteroids were independent risk factors for CPI development. The long-term survival rate of lung cancer patients receiving radiotherapy was not low, but the cumulative incidence of CPI gradually increased to 6.8% at 7 years after radiotherapy. Therefore, close monitoring of CPI development is required in surviving patients with risk factors.