Understanding and managing respiratory infections in children and young adults with neurological impairment.

INTRODUCTION: Patients with neurocognitive impairment (NI) have multiple medical needs, with respiratory problems leading to an important reduction in quality of life and life expectancy. We aimed to explain that the origin of chronic respiratory symptoms in patients with NI is multifactorial. AREAS COVERED: In people with NI there is a high prevalence of swallowing dysfunction and hypersalivation inducing aspiration; cough efficacy is decreased resulting in chronic lung infection; sleep-disordered breathing is frequent and muscle mass is abnormal due to malnutrition. Technical investigations are not always specific and sensitive enough to better diagnose the causes of the respiratory symptoms; moreover, they can sometimes be difficult to perform in this vulnerable patient population. We provide a clinical pathway to adopt to identify, prevent, and treat respiratory complications in children and young adults with NI. A holistic approach in discussion with all care providers and the parents is highly recommended. EXPERT OPINION: The care for people with NI and chronic respiratory problems is challenging. The interplay between several causative factors may be difficult to entangle. Well-performed clinical research in this field is largely missing and should be encouraged. Only then, evidence-based clinical care will become possible for this vulnerable patient group.

Nanomedicine to fight infectious disease.

The ubiquity and potency of antibiotics may give the false impression that infection is a solved problem. Unfortunately, even bacterial infections, the target of antibiotics, remain a major cause of illness and death. Several major unmet needs persist: biofilms, such as those on implanted hardware, largely resist antibiotics; the inflammatory host response to infection often produces more damage than the infection itself; and systemic antibiotics often decimate the gut microbiome, which can predispose to additional infections and even predispose to non-infectious diseases. Additionally, there is an increasing threat from multi-drug resistant microorganisms, though market forces may continue to inhibit innovation in this realm. These numerous unmet infection-related needs provide attractive goals for innovation of targeted drug delivery technologies, especially those of nanomedicine. Here we review several of those innovations in pre-clinical development, the two such therapies which have made it to clinical use, and the opportunities for further technology development for treating infections.

Evaluating the Potential of Ursolic Acid as Bioproduct for Cutaneous and Visceral Leishmaniasis.

Leishmaniasis affects around 12 million people worldwide and is estimated to cause the ninth-largest disease burden. There are three main forms of the disease, visceral (VL), cutaneous (CL), and mucocutaneous (MCL), leading to more than one million new cases every year and several thousand deaths. Current treatments based on chemically synthesized molecules are far from ideal. In this study, we have tested the in vitro and in vivo efficacy of ursolic acid (UA), a multifunctional triterpenoid with well-known antitumoral, antioxidant, and antimicrobial effects on different Leishmania strains. The in vitro antileishmanial activity against the intracellular forms was six and three-fold higher compared to extracellular forms of L. amazonensis and L. infantum, respectively. UA also showed to be a potent antileishmanial drug against both VL and CL manifestations of the disease in experimental models. UA parenterally administered at 5 mg/kg for seven days significantly reduced the parasite burden in liver and spleen not only in murine acute infection but also in a chronic-infection model against L. infantum. In addition, UA ointment (0.2%) topically administered for four weeks diminished (50%) lesion size progression in a chronic infection model of CL caused by L. amazonensis, which was much greater than the effect of UA formulated as an O/W emulsion. UA played a key role in the immunological response modulating the Th1 response. The exposure of Leishmania-infected macrophages to UA led to a significant different production in the cytokine levels depending on the Leishmania strain causing the infection. In conclusion, UA can be a promising therapy against both CL and VL.

Targeting Pseudomonas aeruginosa in the Sputum of Primary Ciliary Dyskinesia Patients with a Combinatorial Strategy Having Antibacterial and Anti-Virulence Potential.

In primary ciliary dyskinesia (PCD) patients, Pseudomonas aeruginosa is a major opportunistic pathogen, frequently involved in chronic infections of the lower airways. Infections by this bacterial species correlates with a worsening clinical prognosis and recalcitrance to currently available therapeutics. The antimicrobial peptide, lin-SB056-1, in combination with the cation chelator ethylenediaminetetraacetic acid (EDTA), was previously demonstrated to be bactericidal against P. aeruginosa in an artificial sputum medium. The purpose of this study was to validate the anti-P. aeruginosa activity of such a combination in PCD sputum and to evaluate the in vitro anti-virulence effects of EDTA. In combination with EDTA, lin-SB056-1 was able to significantly reduce the load of endogenous P. aeruginosa ex vivo in the sputum of PCD patients. In addition, EDTA markedly reduced the production of relevant bacterial virulence factors (e.g., pyocyanin, proteases, LasA) in vitro by two representative mucoid strains of P. aeruginosa isolated from the sputum of PCD patients. These results indicate that the lin-SB056-1/EDTA combination may exert a dual antimicrobial and anti-virulence action against P. aeruginosa, suggesting a therapeutic potential against chronic airway infections sustained by this bacterium.

Targeting Bacterial Biofilms by the Green Tea Polyphenol EGCG.

Bacterial biofilms are multicellular aggregates in which cells are embedded in an extracellular matrix of self-produced biopolymers. Being refractory to antibiotic treatment and host immune systems, biofilms are involved in most chronic infections, and anti-biofilm agents are being searched for urgently. Epigallocatechin-3-gallate (EGCG) was recently shown to act against biofilms by strongly interfering with the assembly of amyloid fibres and the production of phosphoethanolamin-modified cellulose fibrils. Mechanistically, this includes a direct inhibition of the fibre assembly, but also triggers a cell envelope stress response that down-regulates the synthesis of these widely occurring biofilm matrix polymers. Based on its anti-amyloidogenic properties, EGCG seems useful against biofilms involved in cariogenesis or chronic wound infection. However, EGCG seems inefficient against or may even sometimes promote biofilms which rely on other types of matrix polymers, suggesting that searching for 'magic bullet' anti-biofilm agents is an unrealistic goal. Combining molecular and ecophysiological aspects in this review also illustrates why plants control the formation of biofilms on their surfaces by producing anti-amyloidogenic compounds such as EGCG. These agents are not only helpful in combating certain biofilms in chronic infections but even seem effective against the toxic amyloids associated with neuropathological diseases.

Vitamin E Increases Antimicrobial Sensitivity by Inhibiting Bacterial Lipocalin Antibiotic Binding.

Burkholderia cenocepacia is an opportunistic Gram-negative bacterium that causes serious respiratory infections in patients with cystic fibrosis. Recently, we discovered that B. cenocepacia produces the extracellular bacterial lipocalin protein BcnA upon exposure to sublethal concentrations of bactericidal antibiotics. BcnA captures a range of antibiotics outside bacterial cells, providing a global extracellular mechanism of antimicrobial resistance. In this study, we investigated water-soluble and liposoluble forms of vitamin E as inhibitors of antibiotic binding by BcnA. Our results demonstrate that in vitro, both vitamin E forms bind strongly to BcnA and contribute to reduce the MICs of norfloxacin (a fluoroquinolone) and ceftazidime (a ß-lactam), both of them used as model molecules representing two different chemical classes of antibiotics. Expression of BcnA was required for the adjuvant effect of vitamin E. These results were replicated in vivo using the Galleria mellonella larva infection model whereby vitamin E treatment, in combination with norfloxacin, significantly increased larva survival upon infection in a BcnA-dependent manner. Together, our data suggest that vitamin E can be used to increase killing by bactericidal antibiotics through interference with lipocalin binding.IMPORTANCE Bacteria exposed to stress mediated by sublethal antibiotic concentrations respond by adaptive mechanisms leading to an overall increase of antibiotic resistance. One of these mechanisms involves the release of bacterial proteins called lipocalins, which have the ability to sequester antibiotics in the extracellular space before they reach bacterial cells. We speculated that interfering with lipocalin-mediated antibiotic binding could enhance the efficacy of antibiotics to kill bacteria. In this work, we report that when combined with bactericidal antibiotics, vitamin E contributes to enhance bacterial killing both in vitro and in vivo. This adjuvant effect of vitamin E requires the presence of BcnA, a bacterial lipocalin produced by the cystic fibrosis pathogen Burkholderia cenocepacia Since most bacteria produce lipocalins like BcnA, we propose that our findings could be translated into making novel antibiotic adjuvants to potentiate bacterial killing by existing antibiotics.

A High-Throughput Screening Approach To Repurpose FDA-Approved Drugs for Bactericidal Applications against Staphylococcus aureus Small-Colony Variants.

Drug repurposing offers an expedited and economical route to develop new clinical therapeutics in comparison to traditional drug development. Growth-based high-throughput screening is concomitant with drug repurposing and enables rapid identification of new therapeutic uses for investigated drugs; however, this traditional method is not compatible with microorganisms with abnormal growth patterns such as Staphylococcus aureus small-colony variants (SCV). SCV subpopulations are auxotrophic for key compounds in biosynthetic pathways, which result in low growth rate. SCV formation is also associated with reduced antibiotic susceptibility, and the SCV's ability to revert to the normal cell growth state is thought to contribute to recurrence of S. aureus infections. Thus, there is a critical need to identify antimicrobial agents that are potent against SCV in order to effectively treat chronic infections. Accordingly, here we describe adapting an adenylate kinase (AK)-based cell death reporter assay to identify members of a Food and Drug Administration (FDA)-approved drug library that display bactericidal activity against S. aureus SCV. Four library members, daunorubicin, ketoconazole, rifapentine, and sitafloxacin, exhibited potent SCV bactericidal activity against a stable S. aureus SCV. Further investigation showed that sitafloxacin was potent against methicillin-susceptible and -resistant S. aureus, as well as S. aureus within an established biofilm. Taken together, these results demonstrate the ability to use the AK assay to screen small-molecule libraries for SCV bactericidal agents and highlight the therapeutic potential of sitafloxacin to be repurposed to treat chronic S. aureus infections associated with SCV and/or biofilm growth states.IMPORTANCE Conventional antibiotics fail to successfully treat chronic osteomyelitis, endocarditis, and device-related and airway infections. These recurring infections are associated with the emergence of SCV, which are recalcitrant to conventional antibiotics. Studies have investigated antibiotic therapies to treat SCV-related infections but have had little success, emphasizing the need to identify novel antimicrobial drugs. However, drug discovery is a costly and time-consuming process. An alternative strategy is drug repurposing, which could identify FDA-approved and well-characterized drugs that could have off-label utility in treating SCV. In this study, we adapted a high-throughput AK-based assay to identify 4 FDA-approved drugs, daunorubicin, ketoconazole, rifapentine, and sitafloxacin, which display antimicrobial activity against S. aureus SCV, suggesting an avenue for drug repurposing in order to effectively treat SCV-related infections. Additionally, this screening paradigm can easily be adapted for other drug/chemical libraries to identify compounds bactericidal against SCV.

Evaluation of Current and Emerging Antimalarial Medicines for Inhibition of Toxoplasma gondii Growth in Vitro.

Toxoplasma gondii is a common zoonotic infection of humans, and estimates indicate that 1-2 billion people are chronically infected. Although largely asymptomatic, chronic infection poses risk of serious disease due to reactivation should immunity decline. Current therapies for toxoplasmosis only control acute infection caused by actively proliferating tachyzoites but do not eradicate the chronic tissue cyst stages. As well, there are considerable adverse side effects of the most commonly used therapy of combined sulfadiazine and pyrimethamine. Targeting the folate pathway is also an effective treatment for malaria, caused by the related parasites Plasmodium spp., suggesting common agents might be used to treat both infections. Here, we evaluated currently approved and newly emerging medicines for malaria to determine if such compounds might also prove useful for treating toxoplasmosis. Surprisingly, the majority of antimalarial compounds being used currently or in development for treatment of malaria were only modestly effective at inhibiting in vitro growth of T. gondii tachyzoites. These findings suggest that many essential processes in P. falciparum that are targeted by antimalarial compounds are either divergent or nonessential in T. gondii, thus limiting options for repurposing of current antimalarial medicines for toxoplasmosis.

A preliminary investigation on single nucleotide polymorphism rs2287622 of bile salt export pump gene in patients with chronic hepatitis C virus infection in Hunan, China.

BACKGROUND: European researchers have underscored associations between single nucleotide polymorphism (SNP) rs2287622 of the hepatobiliary bile salt export pump (BSEP) gene and the risk of hepatitis C virus (HCV) infection. The distributions of SNP rs2287622 are racially specific. This study was aimed to preliminarily investigate the distribution of BSEP gene SNP rs2287622 in the Han patients with chronic HCV-infection (CHC) in Hunan, China. METHODS: BSEP gene SNP rs2287622 of 165 CHC patients, 99 patients with chronic hepatitis B virus infection (CHB) and 99 healthy individuals were analyzed by polymerase chain reaction-restriction fragment length polymorphism analysis and nucleotide sequencing. RESULTS: The overall frequencies of the C allele of BESP gene SNP rs2287622 in the CHC patients, CHB patients and healthy individuals were 74.2, 72.7 and 74.2%, respectively (P > 0.05). The overall odds ratios (ORs) aiming at predicting CHC risk by comparing the ratios of the frequency distribution of alleles or genotypes in the CHC group with those in the non-CHC group had no statistical significance (P > 0.05). However, the CHC ORs of CC vs TT, TC vs TT and CC + CT vs TT among the individuals aged over 40 years were 2.680, 3.122 and 2.824 respectively (P < 0.05), and the higher risk did not relate to gender, HCV genotypes and presence of HCV-related liver cirrhosis. CONCLUSIONS: Among the Han individuals aged over 40 years in Hunan, China, genotype CC or CT of BSEP gene SNP rs2287622 may correlate with higher risk of CHC in comparison with genotype TT. Further study with a larger cohort is essential.

Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics.

Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 µm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3- as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3- as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment.IMPORTANCEPseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.

Elution of antibiotics from poly(methyl methacrylate) bone cement after extended implantation does not necessarily clear the infection despite susceptibility of the clinical isolates.

Chronic orthopedic infections are commonly caused by bacterial biofilms, which are recalcitrant to antibiotic treatment. In many cases, the revision procedure for periprosthetic joint infection or trauma cases includes the implantation of antibiotic-loaded bone cement to kill infecting bacteria via the elution of a strong local dose of antibiotic(s) at the site. While many studies have addressed the elution kinetics of both non-absorbable and absorbable bone cements both in vitro and in vivo, the potency of ALBC against pathogenic bacteria after extended implantation time is not clear. In this communication, we use two case studies, a Viridans streptococci infected total knee arthroplasty (TKA) and a MRSA-polymicrobial osteomyelitis of a distal tibial traumatic amputation (TA) to demonstrate that an antibiotic-loaded poly(methyl methacrylate) (ALPMMA) coated intermedullary rod implanted for 117 days (TKA) and three ALPMMA suture-strung beads implanted for 210 days (TA) retained killing ability against Pseudomonas aeruginosa and Staphylococcus aureus in vitro, despite different clinical efficacies. The TKA infection resolved and the patient progressed to an uneventful second stage. However, the TA infection only resolved after multiple rounds of debridement, IV vancomycin and removal of the PMMA beads and placement of vancomycin and tobramycin loaded calcium sulfate beads.

Increased risk of intracerebral hemorrhage among patients with chronic osteomyelitis.

OBJECT: Inflammation may provoke cerebral arteriolar ectasia, inducing microaneurysm formation and further promoting intracerebral hemorrhage (ICH). Chronic osteomyelitis (COM) is an inflammatory disorder for which study of its role in ICH is lacking. This study explored whether COM increases the risk of ICH. METHODS: From Taiwan national insurance inpatient claims, 22,052 patients who were newly diagnosed with COM between 1997 and 2010 were identified; 88, 207 age and sex frequency-matched subjects without COM were selected at random for comparison. Risks of ICH associated with COM and comorbidities, including hypertension, diabetes, hyperlipidemia, chronic kidney disease, and drug abuse, were assessed by the end of 2010. RESULTS: The incidence of ICH was 1.68 times higher in the COM cohort than in the comparison cohort, with an adjusted hazard ratio (HR) of 1.50 (95% CI 1.29-1.74) estimated in the multivariable Cox model. Age-specific analysis showed that the HR of ICH for COM patients decreased with age, with an adjusted HR of 3.28 (95% CI 1.88-5.75) in the < 40-year age group, which declined to 1.11 (95% CI 0.88-1.40) in the elderly. The incidence of ICH increased with the severity of COM; for those with severe COM the adjusted HR was 4.42 (95% CI 3.31-5.89). For subjects without comorbidities, the incidence of ICH was 1.20-fold (95% CI 1.00-1.45) higher in the COM cohort than in the comparison cohort. CONCLUSIONS: This study suggests for the first time that COM is an inflammatory factor associated with increased risk of ICH, especially in younger patients.

Safety and immunogenicity of therapeutic DNA vaccine with antiviral drug in chronic HBV patients and its immunogenicity in mice.

BACKGROUND & AIMS: Here, we evaluated the safety and immunogenicity of hepatitis B virus (HBV) DNA vaccine, HB-110, in mice and Korean patients with chronic hepatitis B (CHB) undergoing adefovir dipivoxil (ADV) treatment. METHODS: For animal study, mice (BALB/c or HBV transgenic) were immunized with mHB-110, and T-cell and antibody responses were evaluated. For clinical study, 27 patients randomly received either ADV alone or ADV in combination with HB-110. Liver function tests, serum HBV DNA levels and the presence of HBeAg/anti-HBe were analysed. T-cell responses were estimated by ELISPOT and FACS analysis. RESULTS: mHB-110 induced higher T-cell and antibody responses than mHB-100 in mice. No adverse effects were observed by HB-110 cotreated with ADV. HBV-specific T-cell responses were induced in a portion of patients in medium to high dose of HB-110. Interestingly, HB-110 exhibited positive effects on ALT normalization and maintenance of HBeAg seroconversion. One patient, who received high dose of HB-110 exhibited HBeAg seroconversion during vaccination, which correlated with vaccine-induced T-cell responses without ALT elevation. CONCLUSIONS: HB-110 was safe and tolerable in CHB patients. In contrast to results in animal models, HB-110 in Korean patients exhibited weaker capability of inducing HBV-specific T-cell responses and HBeAg seroconversion than HB-100 in Caucasian patients. As Asian patients, who are generally infected via vertical transmission, appeared to have higher level of immune tolerance than Caucasian, novel approaches for breaking immune tolerance rather than enhancing immunogenicity may be more urgently demanded to develop effective therapeutic HBV DNA vaccines.

Effectiveness of bacteriophages in the sputum of cystic fibrosis patients.

Bacteriophages have been shown to be effective for treating acute infections of the respiratory tract caused by antibiotic-resistant bacteria in animal models, but no evidence has yet been presented of their activity against pathogens in complex biological samples from chronically infected patients. We assessed the efficacy of a cocktail of ten bacteriophages infecting Pseudomonas aeruginosa following its addition to 58 sputum samples from cystic fibrosis (CF) patients collected at three different hospitals. Ten samples that did not contain P. aeruginosa were not analysed further. In the remaining 48 samples, the addition of bacteriophages led to a significant decrease in the levels of P. aeruginosa strains, as shown by comparison with controls, taking two variables (time and bacteriophages) into account (p = 0.024). In 45.8% of these samples, this decrease was accompanied by an increase in the number of bacteriophages. We also tested each of the ten bacteriophages individually against 20 colonies from each of these 48 samples and detected bacteriophage-susceptible bacteria in 64.6% of the samples. An analysis of the clinical data revealed no correlation between patient age, sex, duration of P. aeruginosa colonization, antibiotic treatment, FEV1 (forced expiratory volume in the first second) and the efficacy of bacteriophages. The demonstration that bacteriophages infect their bacterial hosts in the sputum environment, regardless of the clinical characteristics of the patients, represents a major step towards the development of bacteriophage therapy to treat chronic lung infections.