The N-acetylglucosaminidase LytB of Streptococcus pneumoniae is involved in nasopharyngeal colonization and is responsible for cell separation at the end of cell division; thus, lytB mutants form long chains of cells. This paper reports the construction and properties of a defective pneumococcal mutant producing an inactive LytB protein (LytB_E585A). It is shown that an enzymatically active LytB is required for in vitro biofilm formation, as lytB mutants (either lytB or producing the inactive LytB_E585A) are incapable of forming substantial biofilms, despite that extracellular DNA is present in the biofilm matrix. Adding small amounts (0.5 to 2.0 μg/ml) of exogenous LytB or some LytB constructs restored the biofilm-forming capacity of lytB mutants to wild-type levels. The LytB_E585A mutant formed biofilm more rapidly than lytB mutants in the presence of LytB. This suggests that the mutant protein acted in a structural role, likely through the formation of complexes with extracellular DNA. The chain-dispersing capacity of LytB allowed the separation of daughter cells, presumably facilitating the formation of microcolonies and, finally, of biofilms. A role for the possible involvement of LytB in the synthesis of the extracellular polysaccharide component of the biofilm matrix is also discussed.

IMPORTANCE It has been previously accepted that biofilm formation in S. pneumoniae must be a multigenic trait because the mutation of a single gene has led to only to partial inhibition of biofilm production. In the present study, however, evidence that the N-acetylglucosaminidase LytB is crucial in biofilm formation is provided. Despite the presence of extracellular DNA, strains either deficient in LytB or producing a defective LytB enzyme formed only shallow biofilms.

Biofilm formation by Pseudomonas aeruginosa contributes to its survival on surfaces and represents a major clinical threat because of the increased tolerance of biofilms to disinfecting agents. This study aimed to investigate the efficacy of 405-nm light-emitting diode (LED) illumination in eliminating P. aeruginosa biofilms formed on stainless steel coupons under different temperatures. Time-dependent killing assays using planktonic and biofilm cells were used to determine the antimicrobial and antibiofilm activities of LED illumination. We also evaluated the effects of LED illumination on the disinfectant susceptibility, biofilm structure, extracellular polymeric substance (EPS) structure and composition, and biofilm-related gene expression of P. aeruginosa biofilm cells. Results showed that the abundance of planktonic P. aeruginosa cells was reduced by 0.88, 0.53, and 0.85 log CFU/ml following LED treatment for 2 h compared with untreated controls at 4, 10, and 25°C, respectively. For cells in biofilms, significant reductions (1.73, 1.59, and 1.68 log CFU/cm²) were observed following LED illumination for 2 h at 4, 10, and 25°C, respectively. Moreover, illuminated P. aeruginosa biofilm cells were more sensitive to benzalkonium chloride or chlorhexidine than untreated cells. Scanning electron microscopy and confocal laser scanning microscopic observation indicated that both the biofilm structure and EPS structure were disrupted by LED illumination. Further, reverse transcription-quantitative PCR revealed that LED illumination downregulated the transcription of several genes associated with biofilm formation. These findings suggest that LED illumination has the potential to be developed as an alternative method for prevention and control of P. aeruginosa biofilm contamination.

IMPORTANCE Pseudomonas aeruginosa can form biofilms on medical implants, industrial equipment, and domestic surfaces, contributing to high morbidity and mortality rates. This study examined the antibiofilm activity of 405-nm light-emitting diode (LED) illumination against mature biofilms formed on stainless steel coupons. We found that the disinfectant susceptibility, biofilm structure, and extracellular polymeric substance structure and composition were disrupted by LED illumination. We then investigated the transcription of several critical P. aeruginosa biofilm-related genes and analyzed the effect of illumination temperature on the above characteristics. Our results confirmed that LED illumination could be developed into an effective and safe method to counter P. aeruginosa biofilm contamination. Further research will be focused on the efficacy and application of LED illumination for elimination of complicated biofilms in the environment.

Background:

Historically, some chiropractors have been critical of vaccination, and this has been the subject of recent media attention in Canada. We explored the association between media attention and public dissemination of vaccination information on Canadian chiropractors’ websites.

Methods:

In 2016, we identified all Canadian chiropractors’ websites that provided information on vaccination by extracting details from the regulatory college website for each province using the search engine on their "find a chiropractor" page. We assessed the quality of information using the Web Resource Rating Tool (scores range from 0% [worst] to 100% [best]), determined whether vaccination was portrayed in a positive, neutral or negative manner, and conducted thematic analysis of vaccination content. We revisited all identified websites in 2019 to explore for changes to posted vaccination material.

Results:

In July 2016, of 3733 chiropractic websites identified, 94 unique websites provided information on vaccination: 59 (63%) gave negative messaging, 19 (20%) were neutral and 16 (17%) were positive. The quality of vaccination content on the websites was generally poor, with a median Web Resource Rating Tool score of 19%. We identified 4 main themes: there are alternatives to vaccination, vaccines are harmful, evidence regarding vaccination and health policy regarding vaccination. From 2012 to 2016, there was 1 Canadian newspaper story concerning antivaccination statements by chiropractors, whereas 51 news articles were published on this topic between 2017 and 2019. In April 2019, 45 (48%) of the 94 websites we had identified in 2016 had removed all vaccination content or had been discontinued.

Interpretation:

In 2016, a minority of Canadian chiropractors provided vaccination information on their websites, the majority of which portrayed vaccination negatively. After substantial national media attention, about half of all vaccination material on chiropractors’ websites was removed within several years.

Background:

It is unclear how patient-reported access to primary care differs by physician payment model and participation in team-based care. We examined the association between timely and after-hours access to primary care and physician payment model and participation in team-based care, and sought to assess how access varied by patient characteristics.

Methods:

We conducted a cross-sectional analysis of adult (age ≥ 16 yr) Ontarians who responded to the Ontario Health Care Experience Survey between January 2013 and September 2015, reported having a primary care provider and agreed to have their responses linked to health administrative data. Access measures included the proportion of respondents who reported same-day or next-day access when sick, satisfaction with time to appointment when sick, telephone access and knowledge of an after-hours clinic. We tested the association between practice model and measures of access using logistic regression after stratifying for rurality.

Results:

A total of 33 665 respondents met our inclusion criteria. In big cities, respondents in team and nonteam capitation models were less likely to report same-day or next-day access when sick than respondents in enhanced fee-for-service models (team capitation 43%, adjusted odds ratio [OR] 0.88, 95% confidence interval [CI] 0.79–0.98; nonteam capitation 39%, adjusted OR 0.78, 95% CI 0.70–0.87; enhanced fee-for-service 46% [reference]). Respondents in team and nonteam capitation models were more likely than those in enhanced fee-for-service models to report that their provider had an after-hours clinic (team capitation 59%, adjusted OR 2.59, 95% CI 2.39–2.81; nonteam capitation 51%, adjusted OR 1.90, 95% CI 1.76–2.04; enhanced fee-for service 34% [reference]). Patterns were similar for respondents in small towns. There was minimal to no difference by model for satisfaction with time to appointment or telephone access.

Interpretation:

In our setting, there was an association between some types of access to primary care and physician payment model and team-based care, but the direction was not consistent. Different measures of timely access are needed to understand health care system performance.

Background:

Incentive payments for chronic diseases in British Columbia were intended to support primary care physicians in providing more comprehensive care, but research shows that not all physicians bill incentives and not all eligible patients have them billed on their behalf. We investigated patient and physician characteristics associated with billing incentives for chronic diseases in BC.

Methods:

We conducted a retrospective cohort analysis using linked administrative health data to examine community-based primary care physicians and patients with eligible chronic conditions in BC during 2010–2013. Descriptive analyses of patients and physicians compared 3 groups: no incentives in any of the 4 years, incentives in all 4 years, and incentives in any of the study years. We used hierarchical logistic regression models to identify the patient- and physician-level characteristics associated with billing incentives.

Results:

Of 428 770 eligible patients, 142 475 (33.2%) had an incentive billed on their behalf in all 4 years, and 152 686 (35.6%) never did. Of 3936 physicians, 2625 (66.7%) billed at least 1 incentive in each of the 4 years, and 740 (18.8%) billed no incentives during the study period. The strongest predictors of having an incentive billed were the number of physician contacts a patient had (odds ratio [OR] for > 48 contacts 134.77, 95% confidence interval [CI] 112.27–161.78) and whether a physician had a large number of patients in his or her practice for whom incentives were billed (OR 42.38 [95% CI 34.55–52.00] for quartile 4 v. quartile 1).

Interpretation:

The findings suggest that primary care physicians bill incentives for patients based on whom they see most often rather than using a population health management approach to their practice.

Background:

First Nations people in Ontario have an increased prevalence of diabetes compared to other people in the province. This study examined use of health care services by First Nations people with diabetes and other people with diabetes in Ontario.

Methods:

Using linked health administrative databases, we identified all people in Ontario with diabetes as of Apr. 1, 2014. We identified First Nations people using the Indian Register. We looked at outcomes from Apr. 1, 2014, to Mar. 31, 2015. We determined the proportion of people with a regular family physician and their continuity of care with that physician. We also examined visits with specialists for diabetes care, hospital admissions for ambulatory-care–sensitive conditions, and emergency department visits for hypo- or hyperglycemia.

Results:

There were 1 380 529 people diagnosed with diabetes in Ontario as of Apr. 1, 2014, of whom 22 952 (1.7%) were First Nations people. First Nations people were less likely to have a regular family physician (85.3% v. 97.7%) and had lower continuity of care with that physician (mean score for continuity of care 74.6 v. 77.7) than other people in Ontario. They were also less likely to see specialists. First Nations people were more likely to be admitted to hospital for ambulatory-care–sensitive conditions (2.4% v. 1.2%) and to have an emergency department visit for hypo- or hyperglycemia (1.5% v. 0.8%). Disparities were particularly marked for those living in First Nations communities.

Interpretation:

First Nations people with diabetes in Ontario had poorer access to and use of primary care than other people with diabetes in the province. These findings may help explain continued disparities in the rates of complications related to diabetes.

Background:

Peripregnancy emergency department use may be common, but data specific to health care systems like that in Canada are lacking. As prior research was limited to livebirths, omitting pregnancies ending in miscarriage or induced abortion, the current study quantified and characterized emergency department use among women in Ontario with a recognized pregnancy.

Methods:

This retrospective population-based cohort study included all recognized pregnancies among Ontario residents aged 10–55 years with an estimated date of conception between Apr. 1, 2002, and Mar. 31, 2017. We defined peripregnancy emergency department use as any emergency department visit during pregnancy or within 42 days after pregnancy. We used modified Poisson regression with a robust error variance to generate relative risks (RRs) and 95% confidence intervals (CIs) for the outcome of any peripregnancy emergency department use in association with maternal age, parity, residential income quintile, location of residence, immigrant status, antenatal care provider and number of comorbidities within 120 days before the clinical start of the pregnancy (expressed as total number of Aggregated Diagnosis Groups [ADGs] obtained with the Johns Hopkins Adjusted Clinical Group System). All RRs, except for number of comorbidities, were further adjusted for number of ADGs.

Results:

Peripregnancy emergency department use occurred in 1 075 991 (39.4%) of 2 728 236 recognized pregnancies, including 35.8% of livebirths, 47.3% of stillbirths, 73.7% of miscarriages and 84.8% of threatened abortions. A peripregnancy emergency department visit was more likely among women who were less than 25 years of age (adjusted RR 1.16, 95% CI 1.16–1.17), were nulliparous (adjusted RR 1.13, 95% CI 1.13–1.13), resided in the lowest income quintile area (adjusted RR 1.16, 95% CI 1.15–1.16) or in a rural area (adjusted RR 1.50, 95% CI 1.50–1.51), were Canadian-born (adjusted RR 1.22, 95% CI 1.22–1.23), were not seen by an obstetrician (adjusted RR 1.66, 95% CI 1.54–1.80) or had a greater number of ADGs. Emergency department use peaked in the first trimester and in the first week postpartum. Compared to women residing in urban areas, those residing in rural areas had an odds ratio (OR) of 3.44 (95% CI 3.39–3.49) for 3 or more emergency department visits. Women with 3–4 (OR 1.99, 95% CI 1.97–2.01), 5–6 (OR 3.55, 95% CI 3.49–3.61), or 7 or more (OR 7.59, 95% CI 7.39–7.78) prepregnancy comorbidities were more likely to have 3 or more peripregnancy emergency department visits than were those with 2 or fewer comorbidities.

Interpretation:

Peripregnancy emergency department use occurred in nearly 40% of pregnancies, notably in the first trimester and early in the postpartum period. Efforts are needed to streamline rapid access to ambulatory obstetric care during these peak periods, when women are susceptible to miscarriage or a complication after a livebirth.

Background:

In Canada, First Nations populations experience a higher incidence of diabetes and diabetes-related complications than other people. Given the paucity of information on use of preventive eye examinations and the need for interventional care for severe retinopathy among First Nations people, we carried out a population-based study to compare rates of eye examinations and interventional therapies to treat vision-threatening stages of diabetic retinopathy among First Nations people and other people with diabetes in Ontario.

Methods:

In collaboration with the Chiefs of Ontario, we carried out a population-based study to identify cohorts of First Nations people and other people with diabetes in Ontario from 1995/96 to 2014/15. We used linked health administrative databases to evaluate rates of eye examination (2005/06–2014/15) and severe diabetic retinopathy treatment and compared them between the 2 populations, and between First Nations people living in and outside of First Nations communities.

Results:

We identified 23 013 First Nations people and 1 364 222 other people diagnosed with diabetes from 1995/96 to 2014/15, of whom 49.8% (95% confidence interval [CI] 48.9%–50.7%) and 53.8% (95% CI 53.7%–54.0%), respectively, received an eye examination in 2014/15. Eye examination rates were similar for First Nations people regardless of whether they lived in or outside a First Nations community. First Nations people developed severe diabetic retinopathy at a faster rate than other people (hazard ratio 1.19, 95% CI 1.02–1.38). The gap between First Nations people and other people in the proportion requiring therapy for severe diabetic retinopathy was especially prominent among younger people. There were no significant differences in rates of diabetic retinopathy treatment in First Nations people stratified by place of residence.

Interpretation:

Eye examination rates remain suboptimal among people with diabetes in Ontario and were lower among First Nations people. This is particularly concerning in light of our other findings showing an increased risk of requiring treatment for advanced diabetic retinopathy and the accelerated rate of diabetic retinopathy progression among First Nations people with diabetes.

Whether low plasma 25-hydroxyvitamin D concentrations cause osteoporotic fractures is unclear. We tested the hypothesis that low plasma 25-hydroxyvitamin D concentrations are associated with increased risk of osteoporotic fractures using a Mendelian randomization analysis.We genotyped 116 335 randomly chosen white Danish persons aged 20–100 years in 2 population-based cohort studies for plasma 25-hydroxyvitamin D decreasing genotypes in CYP2R1 (rs117913124 and rs12794714), DHCR7 (rs7944926 and rs11234027), GEMIN2 (rs2277458), and HAL (rs3819817); 35 833 had information on plasma 25-hydroxyvitamin D. We assessed risk of total, osteoporotic, and anatomically localized fractures from 1981 through 2017. Information on fractures and vital status was obtained from nationwide registries.During up to 36 years of follow-up, we observed 17 820 total fractures, 10 861 osteoporotic fractures, and 3472 fractures of hip or femur. Compared with individuals with 25-hydroxyvitamin D ≥ 50nmol/L, multivariable adjusted hazard ratios (95% CIs) for total fractures were 1.03 (0.97–1.09) for individuals with 25–49.9 nmol/L, 1.19 (1.10–1.28) for individuals with 12.5–24.9 nmol/L, and 1.39 (1.21–1.60) for individuals with 25-hydroxyvitamin D < 12.5 nmol/L. Corresponding hazard ratios were 1.07 (1.00–1.15), 1.25 (1.13–1.37), and 1.49 (1.25–1.77) for osteoporotic fractures and 1.09 (0.98–1.22), 1.37 (1.18–1.57), and 1.41 (1.09–1.81) for fractures of hip or femur, respectively. Hazard ratios per 1 increase in vitamin D allele score, corresponding to 3.0% (approximately 1.6 nmol/L) lower 25-hydroxyvitamin D concentrations, were 0.99 (0.98–1.00) for total fractures, 0.99 (0.97–1.00) for osteoporotic fractures, and 0.98 (0.95–1.00) for fractures of hip or femur.Low plasma 25-hydroxyvitamin D concentrations were associated with osteoporotic fractures; however, Mendelian randomization analysis provided no evidence supporting a causal role for vitamin D in the risk for osteoporotic fractures.

With increased interest in lipoprotein(a) (Lp[a]) concentration as a target for risk reduction and growing clinical evidence of its impact on cardiovascular disease (CVD) risk, rigorous analytical performance specifications (APS) and accuracy targets for Lp(a) are required. We investigated the biological variation (BV) of Lp(a), and 2 other major biomarkers of CVD, apolipoprotein A-I (apoA-I) and apolipoprotein B-100 (apoB), in the European Biological Variation Study population.Serum samples were drawn from 91 healthy individuals for 10 consecutive weeks at 6 European laboratories and analyzed in duplicate on a Roche Cobas 8000 c702. Outlier, homogeneity, and trend analysis were performed, followed by CV-ANOVA to determine BV estimates and their 95% CIs. These estimates were used to calculate APS and reference change values. For Lp(a), BV estimates were determined on normalized concentration quintiles.Within-subject BV estimates were significantly different between sexes for Lp(a) and between women aged <50 and >50 years for apoA-I and apoB. Lp(a) APS was constant across concentration quintiles and, overall, lower than APS based on currently published data, whereas results were similar for apoA-I and apoB.Using a fully Biological Variation Data Critical Appraisal Checklist (BIVAC)–compliant protocol, our study data confirm BV estimates of Lp(a) listed in the European Federation of Clinical Chemistry and Laboratory Medicine database and reinforce concerns expressed in recent articles regarding the suitability of older APS recommendations for Lp(a) measurements. Given the heterogeneity of Lp(a), more BIVAC-compliant studies on large numbers of individuals of different ethnic groups would be desirable.

The aim of this study was to assess the association of high-sensitivity cardiac troponin (hs-cTnT) and other cardiac, kidney, hyperglycemia, and inflammatory biomarkers with peripheral neuropathy (PN) in a community-based population.We conducted a cross-sectional analysis of 3056 black and white participants in the Atherosclerosis Risk in Communities (ARIC) study who underwent standardized monofilament PN testing and had measures of cardiac function (hs-cTnT, N-terminal pro–B-type natriuretic peptide [NT-proBNP], and growth differentiation factor 15 [GDF15]), kidney function (serum creatinine, cystatin C, β-2 microglobulin, urine albumin-to-creatinine ratio), hyperglycemia (fasting glucose, hemoglobin A_1c [Hb A_1c], fructosamine, glycated albumin, 1,5-anhydroglucitol), and inflammation (C-reactive protein) assessed at visit 6 (2016–2017; age 71–94 years). We used logistic regression to assess the associations of these biomarkers (modeled in diabetes-specific tertiles) with PN in older adults with and without diabetes after adjusting for traditional risk factors.In total, 33.5% of participants had PN (37.3% with diabetes and 31.9% without diabetes). There was an independent association of hs-cTnT with PN regardless of diabetes status (diabetes T3 vs. T1: odds ratio [OR], 2.15 [95% CI, 1.44–3.22]; no diabetes: OR, 2.31 [95%CI, 1.76–3.03]; P = 0.72 for interaction). Among participants without diabetes, there were also significant associations of NT-proBNP (OR, 1.40 [95% CI, 1.08–1.81]) and urine albumin-to-creatinine ratio (OR, 1.55 [95% CI, 1.22–1.97]) with PN. Associations of hyperglycemia biomarkers including Hb A_1c (OR, 1.76 [95% CI, 1.22–2.54]), fructosamine (OR, 1.71 [95% CI, 1.19–2.46]), and glycated albumin (OR, 1.45 [95% CI, 1.03–2.03]) with PN were significant only among participants with diabetes.Overall, hs-cTnT appears to be a global marker of end organ damage, including PN. Laboratory biomarkers may be able to help us identify those individuals with PN.

S100A8/A9 is implicated in inflammation mechanisms related to atherosclerosis and plaque vulnerability, but it remains unclear whether S100A8/A9 is associated with the prognosis of ischemic stroke. The aim of this study was to investigate these associations in 2 independent multicenter cohorts.Plasma S100A8/A9 concentrations at baseline were measured among 4785 patients with ischemic stroke from 2 independent cohorts: Infectious Factors, Inflammatory Markers, and Prognosis of Acute Ischemic Stroke (IIPAIS) and China Antihypertensive Trial in Acute Ischemic Stroke (CATIS). The primary outcome was a composite outcome of death or major disability at 3 months after ischemic stroke. Secondary outcomes were major disability, death, and a composite outcome of death or vascular events.Among the combined participants of IIPAIS and CATIS, the adjusted odds ratios associated with the highest quartile of plasma S100A8/A9 were 2.11 (95% CI, 1.66–2.68) for the primary outcome and 1.62 (95% CI, 1.27–2.07) for the secondary outcome of major disability; adjusted hazard ratios were 4.14 (95% CI, 2.10–8.15) for the secondary outcome of death and 2.08 (95% CI, 1.38–3.13) for the composite outcome of death or vascular events. Each SD increase of log-transformed S100A8/A9 was associated with 28% (95% CI, 18%–39%; P < 0.001) increased risk of the primary outcome. Multivariable-adjusted spline regression analyses showed a linear association between plasma S100A8/A9 concentrations and primary outcome (P < 0.001 for linearity). Subgroup analyses further confirmed these associations.High plasma S100A8/A9 concentrations at baseline were independently associated with increased risks of adverse clinical outcomes at 3 months after ischemic stroke, suggesting that S100A8/A9 might have a role as a prognostic marker of ischemic stroke.

The measurement of neuron-specific enolase (NSE) in serum is frequently requested for diagnosis, risk stratification, and treatment monitoring of neuroblastoma (NB) in the pediatric population. However, authoritative clinical practice guidelines advise about the poor diagnostic performance of NSE.We critically appraised the available literature evaluating the diagnostic and prognostic value of NSE in the management of NB, paying special attention to the definition of appropriate threshold levels. In addition, we discuss the interfering conditions causing artifactual increases of NSE concentrations in serum and potentially influencing the clinical evaluation of patients with suspected NB.No definitive evidence supports the use of serum NSE for diagnosis and monitoring of NB. The risk of obtaining false-positive NSE results associated with confounders (e.g., sample hemolysis) and other pathophysiologic conditions (e.g., inflammation) is remarkable and hampers the diagnostic value of this test. NSE may be helpful to define the risk of death of patients with NB, mainly in the advanced stages of disease. However, further studies validating currently marketed immunoassays and defining threshold values useful for this scope are warranted.

Cardiovascular (CV) disease remains an important cause of morbidity and mortality for patients with chronic kidney disease (CKD). Although clustering of traditional risk factors with CKD is well recognized, kidney-specific mechanisms are believed to drive the disproportionate burden of CV disease. One perturbation that is frequently observed at high rates in patients with CKD is vascular calcification, which may be a central mediator for an array of CV sequelae. This review summarizes the pathophysiological bases of intimal and medial vascular calcification in CKD, current strategies for diagnosis and management, and posits vascular calcification as a risk marker and therapeutic target.

This study showed that treatment with a therapeutic monoclonal immunoglobulin-G1 antibody against phosphorylcholine on oxidized phospholipids preserves coronary flow reserve and attenuates atherosclerotic inflammation as determined by the uptake of ¹⁸F-fluorodeoxyglucose in atherosclerotic mice. The noninvasive imaging techniques represent translational tools to assess the efficacy of phosphorylcholine-targeted therapy on coronary artery function and atherosclerosis in clinical studies.

The Early Career Members Committee (ECMC) of the European Respiratory Society (ERS) consists of 14 members, one Early Career Member (ECM) representative per assembly. In September 2019, seven recently elected representatives joined the ECMC. An overview is given of the tasks performed by each representative within the ECMC (table 1). In addition, a short summary about the content of these tasks is provided.

A 40-year-old, male non-smoker was diagnosed with asthma 6 years ago. He now presents with a 1-week history of worsening breathlessness with fever, cough, and purulent expectoration. He has had >10 emergency department visits and two admissions to hospital in the last 3 months. At each admission, he received bronchodilators and systemic steroids resulting in rapid improvement within 24 h. However, in the current presentation, the patient has no relief with corticosteroids and bronchodilators. His pulse is 140 per min, respiratory rate is 40 per min, blood pressure is 90/60 mmHg and room air oxygen saturation is 80%. Arterial blood gas (ABG) analysis shows hypercapnic respiratory failure. In view this respiratory failure, the patient is intubated and mechanical ventilation initiated. A chest radiograph is shown in figure 1. The therapy initiated includes bronchodilators, a systemic steroid, antibiotics and supportive care.

Caseum, the central necrotic material of tuberculous lesions, is a reservoir of drug-recalcitrant persisting mycobacteria. Caseum is found in closed nodules and in open cavities connecting with an airway. Several commonly accepted characteristics of caseum were established during the preantibiotic era, when autopsies of deceased tuberculosis (TB) patients were common but methodologies were limited. These pioneering studies generated concepts such as acidic pH, low oxygen tension, and paucity of nutrients being the drivers of nonreplication and persistence in caseum. Here we review widely accepted beliefs about the caseum-specific stress factors thought to trigger the shift of Mycobacterium tuberculosis to drug tolerance. Our current state of knowledge reveals that M. tuberculosis is faced with a lipid-rich diet rather than nutrient deprivation in caseum. Variable caseum pH is seen across lesions, possibly transiently acidic in young lesions but overall near neutral in most mature lesions. Oxygen tension is low in the avascular caseum of closed nodules and high at the cavity surface, and a gradient of decreasing oxygen tension likely forms toward the cavity wall. Since caseum is largely made of infected and necrotized macrophages filled with lipid droplets, the microenvironmental conditions encountered by M. tuberculosis in foamy macrophages and in caseum bear many similarities. While there remain a few knowledge gaps, these findings constitute a solid starting point to develop high-throughput drug discovery assays that combine the right balance of oxygen tension, pH, lipid abundance, and lipid species to model the profound drug tolerance of M. tuberculosis in caseum.

Burkholderia cepacia (formerly Pseudomonas cepacia) was once thought to be a single bacterial species but has expanded to the Burkholderia cepacia complex (Bcc), comprising 24 closely related opportunistic pathogenic species. These bacteria have a widespread environmental distribution, an extraordinary metabolic versatility, a complex genome with three chromosomes, and a high capacity for rapid mutation and adaptation. Additionally, they present an inherent resistance to antibiotics and antiseptics, as well as the abilities to survive under nutrient-limited conditions and to metabolize the organic matter present in oligotrophic aquatic environments, even using certain antimicrobials as carbon sources. These traits constitute the reason that Bcc bacteria are considered feared contaminants of aqueous pharmaceutical and personal care products and the frequent reason behind nonsterile product recalls. Contamination with Bcc has caused numerous nosocomial outbreaks in health care facilities, presenting a health threat, particularly for patients with cystic fibrosis and chronic granulomatous disease and for immunocompromised individuals. This review addresses the role of Bcc bacteria as a potential public health problem, the mechanisms behind their success as contaminants of pharmaceutical products, particularly in the presence of biocides, the difficulties encountered in their detection, and the preventive measures applied during manufacturing processes to control contamination with these objectionable microorganisms. A summary of Bcc-related outbreaks in different clinical settings, due to contamination of diverse types of pharmaceutical products, is provided.

Purpose:

Unleashing the immune system by PD-1 and/or CTLA-4 blockade can cause severe immune-related toxicity necessitating immunosuppressive treatment. Whether immunosuppression for toxicity impacts survival is largely unknown.

Experimental Design:

Using data from the prospective nationwide Dutch Melanoma Treatment Registry (DMTR), we analyzed the association between severe toxicity and overall survival (OS) in 1,250 patients with advanced melanoma who were treated with immune checkpoint inhibitors (ICI) in first line between 2012 and 2017. Furthermore, we analyzed whether toxicity management affected survival in these patients.

Results:

A total of 1,250 patients were included, of whom 589 received anti-PD1 monotherapy, 576 ipilimumab, and 85 combination therapy. A total of 312 patients (25%) developed severe (grade ≥3) toxicity. Patients experiencing severe ICI toxicity had a significantly prolonged survival with a median OS of 23 months compared with 15 months for patients without severe toxicity [hazard ratio (HR_adj) = 0.77; 95% confidence interval (CI), 0.63–0.93]. Among patients experiencing severe toxicity, survival was significantly decreased in patients who received anti-TNF ± steroids for steroid-refractory toxicity compared with patients who were managed with steroids only (HR_adj = 1.61; 95% CI, 1.03–2.51), with a median OS of 17 and 27 months, respectively.

Conclusions:

Patients experiencing severe ICI toxicity have a prolonged OS. However, this survival advantage is abrogated when anti-TNF is administered for steroid-refractory toxicity. Further prospective studies are needed to assess the effect of different immunosuppressive regimens on checkpoint inhibitor efficacy.

See related commentary by Weber and Postow, p. 2085

Purpose:

Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer, which can be effectively controlled by immunotherapy with PD-1/PD-L1 checkpoint inhibitors. However, a significant proportion of patients are characterized by primary therapy resistance. Predictive biomarkers for response to immunotherapy are lacking.

Experimental Design:

We applied Bayesian inference analyses on 41 patients with MCC testing various clinical and biomolecular characteristics to predict treatment response. Further, we performed a comprehensive analysis of tumor tissue–based immunologic parameters including multiplexed immunofluorescence for T-cell activation and differentiation markers, expression of immune-related genes and T-cell receptor (TCR) repertoire analyses in 18 patients, seven objective responders, and 11 nonresponders.

Results:

Bayesian inference analyses demonstrated that among currently discussed biomarkers only unimpaired overall performance status and absence of immunosuppression were associated with response to therapy. However, in responders, a predominance of central memory T cells and expression of genes associated with lymphocyte attraction and activation was evident. In addition, TCR repertoire usage of tumor-infiltrating lymphocytes (TILs) demonstrated low T-cell clonality, but high TCR diversity in responding patients. In nonresponders, terminally differentiated effector T cells with a constrained TCR repertoire prevailed. Sequential analyses of tumor tissue obtained during immunotherapy revealed a more pronounced and diverse clonal expansion of TILs in responders indicating an impaired proliferative capacity among TILs of nonresponders upon checkpoint blockade.

Conclusions:

Our explorative study identified new tumor tissue–based molecular characteristics associated with response to anti–PD-1/PD-L1 therapy in MCC. These observations warrant further investigations in larger patient cohorts to confirm their potential value as predictive markers.

Purpose:

To provide a better understanding of the interplay between the immune system and brain metastases to advance therapeutic options for this life-threatening disease.

Experimental Design:

Tumor-infiltrating lymphocytes (TIL) were quantified by semiautomated whole-slide analysis in brain metastases from 81 lung adenocarcinomas. Multi-color staining enabled phenotyping of TILs (CD3, CD8, and FOXP3) on a single-cell resolution. Molecular determinants of the extent of TILs in brain metastases were analyzed by transcriptomics in a subset of 63 patients. Findings in lung adenocarcinoma brain metastases were related to published multi-omic primary lung adenocarcinoma The Cancer Genome Atlas data (n = 230) and single-cell RNA-sequencing (scRNA-seq) data (n = 52,698).

Results:

TIL numbers within tumor islands was an independent prognostic marker in patients with lung adenocarcinoma brain metastases. Comparative transcriptomics revealed that expression of three surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) was closely associated with TIL numbers. Their expression was not only prognostic in brain metastasis but also in primary lung adenocarcinoma. Correlation with scRNA-seq data revealed that brain metastases with high expression of surfactant genes might originate from tumor cells resembling alveolar type 2 cells. Methylome-based estimation of immune cell fractions in primary lung adenocarcinoma confirmed a positive association between lymphocyte infiltration and surfactant expression. Tumors with a high surfactant expression displayed a transcriptomic profile of an inflammatory microenvironment.

Conclusions:

The expression of surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) defines an inflamed subtype of lung adenocarcinoma brain metastases characterized by high abundance of TILs in close vicinity to tumor cells, a prolonged survival, and a tumor microenvironment which might be more accessible to immunotherapeutic approaches.

Purpose:

Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy.

Experimental Design:

PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3 transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti–PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque.

Results:

We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3 bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell–mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti–PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile.

Conclusions:

These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.

Purpose:

There are several agents in early clinical trials targeting components of the adenosine pathway including A2AR and CD73. The identification of cancers with a significant adenosine drive is critical to understand the potential for these molecules. However, it is challenging to measure tumor adenosine levels at scale, thus novel, clinically tractable biomarkers are needed.

Experimental Design:

We generated a gene expression signature for the adenosine signaling using regulatory networks derived from the literature and validated this in patients. We applied the signature to large cohorts of disease from The Cancer Genome Atlas (TCGA) and cohorts of immune checkpoint inhibitor–treated patients.

Results:

The signature captures baseline adenosine levels in vivo (r² = 0.92, P = 0.018), is reduced after small-molecule inhibition of A2AR in mice (r² = –0.62, P = 0.001) and humans (reduction in 5 of 7 patients, 70%), and is abrogated after A2AR knockout. Analysis of TCGA confirms a negative association between adenosine and overall survival (OS, HR = 0.6, P < 2.2e^–16) as well as progression-free survival (PFS, HR = 0.77, P = 0.0000006). Further, adenosine signaling is associated with reduced OS (HR = 0.47, P < 2.2e^–16) and PFS (HR = 0.65, P = 0.0000002) in CD8⁺ T-cell–infiltrated tumors. Mutation of TGFβ superfamily members is associated with enhanced adenosine signaling and worse OS (HR = 0.43, P < 2.2e^–16). Finally, adenosine signaling is associated with reduced efficacy of anti-PD1 therapy in published cohorts (HR = 0.29, P = 0.00012).

Conclusions:

These data support the adenosine pathway as a mediator of a successful antitumor immune response, demonstrate the prognostic potential of the signature for immunotherapy, and inform patient selection strategies for adenosine pathway modulators currently in development.

Purpose:

The development of leptomeningeal melanoma metastases (LMM) is a rare and devastating complication of the late-stage disease, for which no effective treatments exist. Here, we performed a multi-omics analysis of the cerebrospinal fluid (CSF) from patients with LMM to determine how the leptomeningeal microenvironment shapes the biology and therapeutic responses of melanoma cells.

Experimental Design:

A total of 45 serial CSF samples were collected from 16 patients, 8 of these with confirmed LMM. Of those with LMM, 7 had poor survival (<4 months) and one was an extraordinary responder (still alive with survival >35 months). CSF samples were analyzed by mass spectrometry and incubated with melanoma cells that were subjected to RNA sequencing (RNA-seq) analysis. Functional assays were performed to validate the pathways identified.

Results:

Mass spectrometry analyses showed the CSF of most patients with LMM to be enriched for pathways involved in innate immunity, protease-mediated damage, and IGF-related signaling. All of these were anticorrelated in the extraordinary responder. RNA-seq analysis showed CSF to induce PI3K/AKT, integrin, B-cell activation, S-phase entry, TNFR2, TGFβ, and oxidative stress responses in the melanoma cells. ELISA assays confirmed that TGFβ expression increased in the CSF of patients progressing with LMM. CSF from poorly responding patients conferred tolerance to BRAF inhibitor therapy in apoptosis assays.

Conclusions:

These analyses identified proteomic/transcriptional signatures in the CSF of patients who succumbed to LMM. We further showed that the CSF from patients with LMM has the potential to modulate BRAF inhibitor responses and may contribute to drug resistance.

See related commentary by Glitza Oliva and Tawbi, p. 2083

Purpose:

Human B7-H3 (hB7-H3) is a promising molecular imaging target differentially expressed on the neovasculature of breast cancer and has been validated for preclinical ultrasound (US) imaging with anti–B7-H3-antibody-functionalized microbubbles (MB). However, smaller ligands such as affibodies (ABY) are more suitable for the design of clinical-grade targeted MB.

Experimental Design:

Binding of ABY_B7-H3 was confirmed with soluble and cell-surface B7-H3 by flow cytometry. MB were functionalized with ABY_B7-H3 or anti–B7-H3-antibody (Ab_B7-H3). Control and targeted MB were tested for binding to hB7-H3–expressing cells (MS1_hB7-H3) under shear stress conditions. US imaging was performed with MB_ABY-B7-H3 in an orthotopic mouse model of human MDA-MB-231 coimplanted with MS1_hB7-H3 or control MS1_WT cells and a transgenic mouse model of breast cancer development.

Results:

ABY_B7-H3 specifically binds to MS1_hB7-H3 and murine-B7-H3–expressing monocytes. MB_ABY-B7-H3 (8.5 ± 1.4 MB/cell) and MB_Ab-B7-H3 (9.8 ± 1.3 MB/cell) showed significantly higher (P < 0.0001) binding to the MS1_hB7-H3 cells compared with control MB_Non-targeted (0.5 ± 0.1 MB/cell) under shear stress conditions. In vivo, MB_ABY-B7-H3 produced significantly higher (P < 0.04) imaging signal in orthotopic tumors coengrafted with MS1_hB7-H3 (8.4 ± 3.3 a.u.) compared with tumors with MS1_WT cells (1.4 ± 1.0 a.u.). In the transgenic mouse tumors, MB_ABY-B7-H3 (9.6 ± 2.0 a.u.) produced higher (P < 0.0002) imaging signal compared with MB_Non-targeted (1.3 ± 0.3 a.u.), whereas MB_ABY-B7-H3 signal in normal mammary glands and tumors with B7-H3 blocking significantly reduced (P < 0.02) imaging signal.

Conclusions:

MB_ABY-B7-H3 enhances B7-H3 molecular signal in breast tumors, improving cancer detection, while offering the advantages of a small size ligand and easier production for clinical imaging.

Purpose:

Thymidine kinase 1 (TK1) is downstream to the CDK4/6 pathway, and TK activity (TKa) measured in blood is a dynamic marker of outcome in patients with advanced breast cancer (ABC). This study explores TK1 as a biomarker of palbociclib response, both in vitro and in patients with ABC.

Experimental Design:

Modulation of TK1 levels and activity by palbociclib were studied in seven estrogen receptor–positive breast cancer cell lines: sensitive (PDS) and with palbociclib acquired resistance (PDR). TKa was assayed in plasma obtained at baseline (T0), after one cycle (T1), and at disease progression on palbociclib (T2) in patients enrolled in the "To Reverse ENDocrine Resistance" (TREnd) trial (n = 46).

Results:

Among E2F-dependent genes, TK1 was significantly downregulated after short-term palbociclib. Early TKa reduction by palbociclib occurred in PDS but not in PDR cells. In patients, median TKa (mTKa) at T0 was 75 DiviTum units per liter (Du/L), with baseline TKa not proving prognostic. At T1, mTKa decreased to 35 Du/L, with a minority of patients (n = 8) showing an increase—correlating with a worse outcome than those with decreased/stable TKa (n = 33; mPFS 3.0 vs 9.0 months; P = 0.002). At T2, mTKa was 251 Du/L; patients with TKa above the median had worse outcomes on post-study treatment compared with those with lower TKa (2.9 vs 8.7 months; P = 0.05).

Conclusions:

TK is a dynamic marker of resistance to palbociclib which may lead to early identification of patients in whom treatment escalation may be feasible. In addition, TKa may stratify prognosis in patients with acquired resistance to palbociclib.

Purpose:

KEYNOTE-158 (ClinicalTrials.gov identifier: NCT02628067) investigated the efficacy and safety of pembrolizumab across multiple cancers. We present results from patients with previously treated advanced well-differentiated neuroendocrine tumors (NET).

Patients and Methods:

Pembrolizumab 200 mg was administered every 3 weeks for 2 years or until progression, intolerable toxicity, or physician/patient decision. Tumor imaging was performed every 9 weeks for the first year and then every 12 weeks. Endpoints included objective response rate (ORR) per RECIST v1.1 by independent central radiologic review (primary) and duration of response (DOR), progression-free survival (PFS), overall survival (OS), and safety (secondary).

Results:

A total of 107 patients with NETs of the lung, appendix, small intestine, colon, rectum, or pancreas were treated. Median age was 59.0 years (range, 29–80), 44.9% had ECOG performance status 1, 40.2% had received ≥3 prior therapies for advanced disease, and 15.9% had PD-L1–positive tumors (combined positive score ≥1). Median follow-up was 24.2 months (range, 0.6–33.4). ORR was 3.7% (95% CI, 1.0–9.3), with zero complete responses and four partial responses (three pancreatic and one rectal) all in patients with PD-L1–negative tumors. Median DOR was not reached, with one of four responses ongoing after ≥21 months follow-up. Median PFS was 4.1 months (95% CI, 3.5–5.4); the 6-month PFS rate was 39.3%. Median OS was 24.2 months (95% CI, 15.8–32.5). Treatment-related adverse events (AE) occurred in 75.7% of patients, 21.5% of whom had grade 3–5 AEs.

Conclusions:

Pembrolizumab monotherapy showed limited antitumor activity and manageable safety in patients with previously treated advanced well-differentiated NETs.

Purpose:

Preclinical data demonstrating androgen receptor (AR)–positive (AR⁺) triple-negative breast cancer (TNBC) cells are sensitive to AR antagonists, and PI3K inhibition catalyzed an investigator-initiated, multi-institutional phase Ib/II study TBCRC032. The trial investigated the safety and efficacy of the AR-antagonist enzalutamide alone or in combination with the PI3K inhibitor taselisib in patients with metastatic AR⁺ (≥10%) breast cancer.

Patients and Methods:

Phase Ib patients [estrogen receptor positive (ER⁺) or TNBC] with AR⁺ breast cancer received 160 mg enzalutamide in combination with taselisib to determine dose-limiting toxicities and the maximum tolerated dose (MTD). Phase II TNBC patients were randomized to receive either enzalutamide alone or in combination with 4 mg taselisib until disease progression. Primary endpoint was clinical benefit rate (CBR) at 16 weeks.

Results:

The combination was tolerated, and the MTD was not reached. The adverse events were hyperglycemia and skin rash. Overall, CBR for evaluable patients receiving the combination was 35.7%, and median progression-free survival (PFS) was 3.4 months. Luminal AR (LAR) TNBC subtype patients trended toward better response compared with non-LAR (75.0% vs. 12.5%, P = 0.06), and increased PFS (4.6 vs. 2.0 months, P = 0.082). Genomic analyses revealed subtype-specific treatment response, and novel FGFR2 fusions and AR splice variants.

Conclusions:

The combination of enzalutamide and taselisib increased CBR in TNBC patients with AR⁺ tumors. Correlative analyses suggest AR protein expression alone is insufficient for identifying patients with AR-dependent tumors and knowledge of tumor LAR subtype and AR splice variants may identify patients more or less likely to benefit from AR antagonists.

Purpose:

Radium-223 is approved for metastatic castration-resistant prostate cancer (mCRPC) based on improved overall survival, and delay in skeletal related events. However, it is not associated with PSA or radiographic response, which poses a challenge in real-time assessment of its efficacy. Surrogate markers of treatment outcomes may facilitate tailoring treatment duration with radium-223, by limiting the duration of therapy with radium-223 in these patients. Here, we sought to investigate the utility of bone metabolic markers (BMMs) as surrogate markers of response to radium-223 in mCRPC.

Patients and Methods:

A prospective phase II trial of radium-223 plus enzalutamide (RE) versus enzalutamide alone was designed to assess surrogacy of BMMs with respect to response to radium-223. Enzalutamide was used as a comparator in lieu of placebo due to the progressive disease. Co-primary endpoints were relative change in serum BMM N-telopeptide (NTP) levels from baseline to 6 months between the two arms and safety and feasibility of the combination.

Results:

Thirty-nine men were randomized to RE (n = 27) or enzalutamide (n = 12). Combination was safe and feasible. Primary endpoint was met. A statistically significant relative change to NTP ratios between arms (0.64, 95% confidence interval, 0.51–0.81; P = 0.00048) favored RE versus enzalutamide. Overall, BMMs decreased with the RE therapy compared with enzalutamide. Improved PSA response rate in RE versus enzalutamide (P = 0.024), correlated with decline in BMMs.

Conclusions:

BMMs declined significantly with combination therapy, and were associated with improved outcomes. Upon external validation, BMMs may emerge as surrogate markers to monitor treatment with radium-223 in real-time.

Purpose:

In the phase III DUO trial, duvelisib, an oral dual PI3K-, inhibitor, demonstrated significantly improved efficacy versus ofatumumab [median (m) progression-free survival (PFS), 13.3 vs. 9.9 months (HR, 0.52; P < 0.0001); overall response rate [ORR], 74% vs. 45% (P < 0.0001)], with a manageable safety profile in patients with relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). We report results from patients with progressive disease (PD) after ofatumumab who crossed over to duvelisib in the DUO trial.

Patients and Methods:

Patients with radiographically confirmed PD after ofatumumab received duvelisib 25 mg twice daily in 28-day cycles until PD, intolerance, death, or study withdrawal. The primary endpoint was ORR per investigator. Secondary endpoints included duration of response (DOR), PFS, and safety.

Results:

As of December 14, 2018, 90 ofatumumab-treated patients in the DUO trial prior to crossover had an ORR of 29%, mDOR of 10.4 months, and mPFS of 9.4 months. After crossover, 77% of patients (69/90) achieved a response, with an mDOR of 14.9 months and mPFS of 15.7 months. Patients with del(17p) and/or TP53 mutations had similar outcomes [ORR, 77% (20/26); mPFS, 14.7 months]. Notably, 73% of patients (47/64) with disease previously refractory to ofatumumab achieved a response. The most frequent any-grade/grade 3/4 treatment-emergent adverse events were diarrhea (47%/23%), neutropenia (26%/23%), pyrexia (24%/4%), cutaneous reactions (23%/4%), and thrombocytopenia (10%/6%).

Conclusions:

Duvelisib demonstrated high response rates with good durability and a manageable safety profile in patients with R/R CLL/SLL who progressed on ofatumumab, including patients with high-risk disease and disease previously refractory to ofatumumab.

Recent FDA approvals of regimens targeting programmed death 1 (PD-1) in combination with anti-CTLA-4 or with VEGF tyrosine kinase inhibitors are reshaping front-line therapy for metastatic kidney cancer. In parallel, therapeutics specific for programmed death ligand 1 (PD-L1), one of the two major ligands for PD-1, are under continued investigation. Surprisingly, not all PD-1 and PD-L1 agents lead to similar clinical outcomes, potentially due to biological differences in the cellular expression and regulation of these targets. Here, we review current clinical data on combination immune checkpoint inhibitor therapy in metastatic kidney cancer and discuss the relevant biology of PD-1 and PD-L1. The design of future rational combination therapy trials in metastatic renal cell carcinoma will rely upon an understanding of this biology, along with an evolving understanding of immune cell populations and their functional states in the tumor microenvironment.

A strain of extensively drug-resistant (XDR) Salmonella enterica serovar Typhi has caused a large ongoing outbreak in Pakistan since 2016. In Ontario, Canada, 10 cases of mainly bloodstream infections (n = 9) were identified in patients who traveled to Pakistan. Whole-genome sequencing showed that Canadian cases were genetically related to the Pakistan outbreak strain. The appearance of XDR typhoid cases in Ontario prompted a provincial wide alert to physicians to recommend treatment with carbapenems or azithromycin in suspected typhoid cases with travel history to Pakistan.

Quinolones, such as the antimalarial atovaquone, are inhibitors of the malarial mitochondrial cytochrome bc₁ complex, a target critical to the survival of both liver- and blood-stage parasites, making these drugs useful as both prophylaxis and treatment. Recently, several derivatives of endochin have been optimized to produce novel quinolones that are active in vitro and in animal models. While these quinolones exhibit potent ex vivo activity against Plasmodium falciparum and Plasmodium vivax, their activity against the zoonotic agent Plasmodium knowlesi is unknown. We screened several of these novel endochin-like quinolones (ELQs) for their activity against P. knowlesi in vitro and compared this with their activity against P. falciparum tested under identical conditions. We demonstrated that ELQs are potent against P. knowlesi (50% effective concentration, <117 nM) and equally effective against P. falciparum. We then screened selected quinolones and partner drugs using a longer exposure (2.5 life cycles) and found that proguanil is 10-fold less potent against P. knowlesi than P. falciparum, while the quinolones demonstrate similar potency. Finally, we used isobologram analysis to compare combinations of the ELQs with either proguanil or atovaquone. We show that all quinolone combinations with proguanil are synergistic against P. falciparum. However, against P. knowlesi, no evidence of synergy between proguanil and the quinolones was found. Importantly, the combination of the novel quinolone ELQ-300 with atovaquone was synergistic against both species. Our data identify potentially important species differences in proguanil susceptibility and in the interaction of proguanil with quinolones and support the ongoing development of novel quinolones as potent antimalarials that target multiple species.

Cefazolin has become a prominent therapy for methicillin-susceptible Staphylococcus aureus (MSSA) infections. However, an important concern is the cefazolin inoculum effect (CzIE), a phenomenon mediated by staphylococcal β-lactamases. Four variants of staphylococcal β-lactamases have been described based on serological methodologies and limited sequence information. Here, we sought to reassess the classification of staphylococcal β-lactamases and their correlation with the CzIE. We included a large collection of 690 contemporary bloodstream MSSA isolates recovered from Latin America, a region with a high prevalence of the CzIE. We determined cefazolin MICs at standard and high inoculums by broth microdilution. Whole-genome sequencing was performed to classify the β-lactamase in each isolate based on the predicted full sequence of BlaZ. We used the classical schemes for β-lactamase classification and compared it to BlaZ allotypes found in unique sequences using the genomic information. Phylogenetic analyses were performed based on the BlaZ and core-genome sequences. The overall prevalence of the CzIE was 40%. Among 641 genomes, type C was the most predominant β-lactamase (37%), followed by type A (33%). We found 29 allotypes and 43 different substitutions in BlaZ. A single allotype, designated BlaZ-2, showed a robust and statistically significant association with the CzIE. Two other allotypes (BlaZ-3 and BlaZ-5) were associated with a lack of the CzIE. Three amino acid substitutions (A9V, E112A, and G145E) showed statistically significant association with the CzIE (P = <0.01). CC30 was the predominant clone among isolates displaying the CzIE. Thus, we provide a novel approach to the classification of the staphylococcal β-lactamases with the potential to more accurately identify MSSA strains exhibiting the CzIE.

Tigecycline serves as one of the antibiotics of last resort to treat multidrug-resistant (including carbapenem-resistant) pathogens. However, the recently emerged plasmid-mediated tigecycline resistance mechanism, Tet(X), challenges the clinical efficacy of this class of antibiotics. In this study, we detected 180 tet(X)-harboring Acinetobacter isolates (8.9%, n = 180) from 2,018 samples collected from avian farms and adjacent environments in China. Eighteen tet(X)-harboring isolates (10.0%) were found to cocarry the carbapenemase gene bla_NDM-1, mostly from waterfowl samples (94.4%, 17/18). Interestingly, among six Acinetobacter strains, tet(X) and bla_NDM-1 were found to colocalize on the same plasmids. Moreover, whole-genome sequencing (WGS) revealed a novel orthologue of tet(X) in the six isolates coharboring tet(X) and bla_NDM-1. Inverse PCR suggested that the two tet(X) genes form a single transposable unit and may be cotransferred. Sequence comparison between six tet(X)- and bla_NDM-1-coharboring plasmids showed that they shared a highly homologous plasmid backbone even though they were isolated from different Acinetobacter species (three from Acinetobacter indicus, two from Acinetobacter schindleri, and one from Acinetobacter lwoffii) from various sources and from different geological regions, suggesting the horizontal genetic transfer of a common tet(X)- and bla_NDM-1-coharboring plasmid among Acinetobacter species in China. Emergence and spread of such plasmids and strains are of great clinical concern, and measures must be implemented to avoid their dissemination.

Capsid assembly is a critical step in the hepatitis B virus (HBV) life cycle, mediated by the core protein. Core is a potential target for new antiviral therapies, the capsid assembly modulators (CAMs). JNJ-56136379 (JNJ-6379) is a novel and potent CAM currently in phase II trials. We evaluated the mechanisms of action (MOAs) and antiviral properties of JNJ-6379 in vitro. Size exclusion chromatography and electron microscopy studies demonstrated that JNJ-6379 induced the formation of morphologically intact viral capsids devoid of genomic material (primary MOA). JNJ-6379 accelerated the rate and extent of HBV capsid assembly in vitro. JNJ-6379 specifically and potently inhibited HBV replication; its median 50% effective concentration (EC₅₀) was 54 nM (HepG2.117 cells). In HBV-infected primary human hepatocytes (PHHs), JNJ-6379, when added with the viral inoculum, dose-dependently reduced extracellular HBV DNA levels (median EC₅₀ of 93 nM) and prevented covalently closed circular DNA (cccDNA) formation, leading to a dose-dependent reduction of intracellular HBV RNA levels (median EC₅₀ of 876 nM) and reduced antigen levels (secondary MOA). Adding JNJ-6379 to PHHs 4 or 5 days postinfection reduced extracellular HBV DNA and did not prevent cccDNA formation. Time-of-addition PHH studies revealed that JNJ-6379 most likely interfered with postentry processes. Collectively, these data demonstrate that JNJ-6379 has dual MOAs in the early and late steps of the HBV life cycle, which is different from the MOA of nucleos(t)ide analogues. JNJ-6379 is in development for chronic hepatitis B treatment and may translate into higher HBV functional cure rates.

Nosocomial pneumonia (NP), including ventilator-associated pneumonia (VAP), is increasingly associated with multidrug-resistant Gram-negative pathogens. This study describes the in vitro activity of ceftazidime-avibactam, ceftazidime, and relevant comparator agents against bacterial pathogens isolated from patients with NP, including VAP, enrolled in a ceftazidime-avibactam phase 3 trial. Gram-positive pathogens were included if coisolated with a Gram-negative pathogen. In vitro susceptibility was determined at a central laboratory using Clinical and Laboratory Standards Institute broth microdilution methods. Of 817 randomized patients, 457 (55.9%) had ≥1 Gram-negative bacterial pathogen(s) isolated at baseline, and 149 (18.2%) had ≥1 Gram-positive pathogen(s) coisolated. The most common isolated pathogens were Klebsiella pneumoniae (18.8%), Pseudomonas aeruginosa (15.8%), and Staphylococcus aureus (11.5%). Ceftazidime-avibactam was highly active in vitro against 370 isolates of Enterobacteriaceae, with 98.6% susceptible (MIC₉₀, 0.5 μg/ml) compared with 73.2% susceptible for ceftazidime (MIC₉₀, >64 μg/ml). The percent susceptibility values for ceftazidime-avibactam and ceftazidime against 129 P. aeruginosa isolates were 88.4% and 72.9% (MIC₉₀ values of 16 μg/ml and 64 μg/ml), respectively. Among ceftazidime-nonsusceptible Gram-negative isolates, ceftazidime-avibactam percent susceptibility values were 94.9% for 99 Enterobacteriaceae and 60.0% for 35 P. aeruginosa. MIC₉₀ values for linezolid and vancomycin (permitted per protocol for Gram-positive coverage) were within their respective MIC susceptibility breakpoints against the Gram-positive pathogens isolated. This analysis demonstrates that ceftazidime-avibactam was active in vitro against the majority of Enterobacteriaceae and P. aeruginosa isolates from patients with NP, including VAP, in a phase 3 trial. (This study has been registered at ClinicalTrials.gov under identifier NCT01808092.)