Fnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes (fnr1, fnr3, fnr5, and fnr7) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Both the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to a 50% reduction in nitrogenase activity, and double deletion of fnr1 and fnr3 resulted to a 90% reduction in activity. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic conditions. Fnr1 and Fnr3 inhibited expression of the genes involved in the aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes.

IMPORTANCE The members of the nitrogen-fixing Paenibacillus spp. have great potential to be used as a bacterial fertilizer in agriculture. However, the functions of the fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we found that in P. polymyxa WLY78, Fnr1 and Fnr3 were responsible for regulation of numerous genes in response to changes in oxygen levels, but Fnr5 and Fnr7 exhibited little effect. Fnr1 and Fnr3 indirectly or directly regulated many types of important metabolism, such as nitrogen fixation, Fe uptake, respiration, and electron transport. This study not only reveals the function of the fnr genes of P. polymyxa WLY78 in nitrogen fixation and other metabolisms but also will provide insight into the evolution and regulatory mechanisms of fnr in Paenibacillus.

The Stenotrophomonas maltophilia complex (Smc) comprises opportunistic environmental Gram-negative bacilli responsible for a variety of infections in both humans and animals. Beyond its large genetic diversity, its genetic organization in genogroups was recently confirmed through the whole-genome sequencing of human and environmental strains. As they are poorly represented in these analyses, we sequenced the whole genomes of 93 animal strains to determine their genetic background and characteristics. Combining these data with 81 newly sequenced human strains and the genomes available from RefSeq, we performed a genomic analysis that included 375 nonduplicated genomes with various origins (animal, 104; human, 226; environment, 30; unknown, 15). Phylogenetic analysis and clustering based on genome-wide average nucleotide identity confirmed and specified the genetic organization of Smc in at least 20 genogroups. Two new genogroups were identified, and two previously described groups were further divided into two subgroups each. Comparing the strains isolated from different host types and their genogroup affiliation, we observed a clear disequilibrium in certain groups. Surprisingly, some antimicrobial resistance genes, integrons, and/or clusters of attC sites lacking integron-integrase (CALIN) sequences targeting antimicrobial compounds extensively used in animals were mainly identified in animal strains. We also identified genes commonly found in animal strains coding for efflux systems. The result of a large whole-genome analysis performed by us supports the hypothesis of the putative contribution of animals as a reservoir of Stenotrophomonas maltophilia complex strains and/or resistance genes for strains in humans.

IMPORTANCE Given its naturally large antimicrobial resistance profile, the Stenotrophomonas maltophilia complex (Smc) is a set of emerging pathogens of immunosuppressed and cystic fibrosis patients. As it is group of environmental microorganisms, this adaptation to humans is an opportunity to understand the genetic and metabolic selective mechanisms involved in this process. The previously reported genomic organization was incomplete, as data from animal strains were underrepresented. We added the missing piece of the puzzle with whole-genome sequencing of 93 strains of animal origin. Beyond describing the phylogenetic organization, we confirmed the genetic diversity of the Smc, which could not be estimated through routine phenotype- or matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF)-based laboratory tests. Animals strains seem to play a key role in the diversity of Smc and could act as a reservoir for mobile resistance genes. Some genogroups seem to be associated with particular hosts; the genetic support of this association and the role of the determinants/corresponding genes need to be explored.

Burkholderia sp. strain SG-MS1 and Pseudomonas sp. strain SG-MS2 have previously been found to mineralize (+)-pinoresinol through a common catabolic pathway. Here, we used comparative genomics, proteomics, protein semipurification, and heterologous expression to identify a flavoprotein from the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family in SG-MS2 that carries out the initial hydroxylation of (+)-pinoresinol at the benzylic carbon. The cognate gene is translationally coupled with a downstream cytochrome gene, and the cytochrome is required for activity. The flavoprotein has a unique combination of cofactor binding and cytochrome requirements for the VAO/PCMH family. The heterologously expressed enzyme has a K_m of 1.17 μM for (+)-pinoresinol. The enzyme is overexpressed in strain SG-MS2 upon exposure to (+)-pinoresinol, along with 45 other proteins, 22 of which were found to be encoded by genes in an approximately 35.1-kb cluster also containing the flavoprotein and cytochrome genes. Homologs of 18 of these 22 genes, plus the flavoprotein and cytochrome genes, were also found in a 38.7-kb cluster in SG-MS1. The amino acid identities of four of the other proteins within the SG-MS2 cluster suggest they catalyze conversion of hydroxylated pinoresinol to protocatechuate and 2-methoxyhydroquinone. Nine other proteins upregulated in SG-MS2 on exposure to (+)-pinoresinol appear to be homologs of proteins known to comprise the protocatechuate and 2-methoxyhydroquinone catabolic pathways, but only three of the cognate genes lie within the cluster containing the flavoprotein and cytochrome genes.

IMPORTANCE (+)-Pinoresinol is an important plant defense compound, a major food lignan for humans and some other animals, and the model compound used to study degradation of the β-β' linkages in lignin. We report a gene cluster, in one strain each of Pseudomonas and Burkholderia, that is involved in the oxidative catabolism of (+)-pinoresinol. The flavoprotein component of the α-hydroxylase which heads the pathway belongs to the 4-phenol oxidizing (4PO) subgroup of the vanillyl alcohol oxidase/p-cresol methyl hydroxylase (VAO/PCMH) enzyme family but constitutes a novel combination of cofactor and electron acceptor properties for the family. It is translationally coupled with a cytochrome gene whose product is also required for activity. The work casts new light on the biology of (+)-pinoresinol and its transformation to other bioactive molecules. Potential applications of the findings include new options for deconstructing lignin into useful chemicals and the generation of new phytoestrogenic enterolactones from lignans.

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia coli fadD mutant JW1794. RpfB activates long-chain fatty acids (n-C_16:0 and n-C_18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes. Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2. In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenes.

IMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes. Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes. Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes. Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

Deep-subsurface hot brines in northwest Poland, extracted through boreholes reaching 1.6 and 2.6 km below the ground surface, were microbiologically investigated using culture-independent and culture-dependent methods. The high-throughput sequencing of 16S rRNA gene amplicons showed a very low diversity of bacterial communities, which were dominated by phyla Proteobacteria and Firmicutes. Bacterial genera potentially involved in sulfur oxidation and nitrate reduction (Halothiobacillus and Methylobacterium) prevailed in both waters over the sulfate reducers ("Candidatus Desulforudis" and Desulfotomaculum). Only one archaeal taxon, affiliated with the order Thermoplasmatales, was detected in analyzed samples. Bacterial isolates obtained from these deep hot brines were closely related to Bacillus paralicheniformis based on the 16S rRNA sequence similarity. However, genomic and physiological analyses made for one of the isolates, Bacillus paralicheniformis strain TS6, revealed the existence of more diverse metabolic pathways than those of its moderate-temperature counterpart. These specific traits may be associated with the ecological adaptations to the extreme habitat, which suggest that some lineages of B. paralicheniformis are halothermophilic.

IMPORTANCE Deep-subsurface aquifers, buried thousands of meters down the Earth’s crust, belong to the most underexplored microbial habitats. Although a few studies revealed the existence of microbial life at the depths, the knowledge about the microbial life in the deep hydrosphere is still scarce due to the limited access to such environments. Studying the subsurface microbiome provides unique information on microbial diversity, community structure, and geomicrobiological processes occurring under extreme conditions of the deep subsurface. Our study shows that low-diversity microbial assemblages in subsurface hot brines were dominated by the bacteria involved in biogeochemical cycles of sulfur and nitrogen. Based on genomic and physiological analyses, we found that the Bacillus paralicheniformis isolate obtained from the brine under study differed from the mesophilic species in the presence of specific adaptations to harsh environmental conditions. We indicate that some lineages of B. paralicheniformis are halothermophilic, which was not previously reported.

Biological nitrogen fixation can be an important source of nitrogen in tropical forests that serve as a major CO₂ sink. Extensive deforestation of the Amazon is known to influence microbial communities and the biogeochemical cycles they mediate. However, it is unknown how diazotrophs (nitrogen-fixing microorganisms) respond to deforestation and subsequent ecosystem conversion to agriculture, as well as whether they can recover in secondary forests that are established after agriculture is abandoned. To address these knowledge gaps, we combined a spatially explicit sampling approach with high-throughput sequencing of nifH genes. The main objectives were to assess the functional distance decay relationship of the diazotrophic bacterial community in a tropical forest ecosystem and to quantify the roles of various factors that drive the observed changes in the diazotrophic community structure. We observed an increase in local diazotrophic diversity (α-diversity) with a decrease in community turnover (β-diversity), associated with a shift in diazotrophic community structure as a result of the forest-to-pasture conversion. Both diazotrophic community turnover and structure showed signs of recovery in secondary forests. Changes in the diazotrophic community were primarily driven by the change in land use rather than differences in geochemical characteristics or geographic distances. The diazotroph communities in secondary forests resembled those in primary forests, suggesting that at least partial recovery of diazotrophs is possible following agricultural abandonment.

IMPORTANCE The Amazon region is a major tropical forest region that is being deforested at an alarming rate to create space for cattle ranching and agriculture. Diazotrophs (nitrogen-fixing microorganisms) play an important role in supplying soil N for plant growth in tropical forests. It is unknown how diazotrophs respond to deforestation and whether they can recover in secondary forests that establish after agriculture is abandoned. Using high-throughput sequencing of nifH genes, we characterized the response of diazotrophs’ β-diversity and identified major drivers of changes in diazotrophs from forest-to-pasture and pasture-to-secondary-forest conversions. Studying the impact of land use change on diazotrophs is important for a better understanding of the impact of deforestation on tropical forest ecosystem functioning, and our results on the potential recovery of diazotrophs in secondary forests imply the possible restoration of ecosystem functions in secondary forests.

Most freshwater bacterial communities are characterized by a few dominant taxa that are often ubiquitous across freshwater biomes worldwide. Our understanding of the genomic diversity within these taxonomic groups is limited to a subset of taxa. Here, we investigated the genomic diversity that enables Limnohabitans, a freshwater genus key in funneling carbon from primary producers to higher trophic levels, to achieve abundance and ubiquity. We reconstructed eight putative Limnohabitans metagenome-assembled genomes (MAGs) from stations located along broad environmental gradients existing in Lake Michigan, part of Earth’s largest surface freshwater system. De novo strain inference analysis resolved a total of 23 strains from these MAGs, which strongly partitioned into two habitat-specific clusters with cooccurring strains from different lineages. The largest number of strains belonged to the abundant LimB lineage, for which robust in situ strain delineation had not previously been achieved. Our data show that temperature and nutrient levels may be important environmental parameters associated with microdiversification within the Limnohabitans genus. In addition, strains predominant in low- and high-phosphorus conditions had larger genomic divergence than strains abundant under different temperatures. Comparative genomics and gene expression analysis yielded evidence for the ability of LimB populations to exhibit cellular motility and chemotaxis, a phenotype not yet associated with available Limnohabitans isolates. Our findings broaden historical marker gene-based surveys of Limnohabitans microdiversification and provide in situ evidence of genome diversity and its functional implications across freshwater gradients.

IMPORTANCE Limnohabitans is an important bacterial taxonomic group for cycling carbon in freshwater ecosystems worldwide. Here, we examined the genomic diversity of different Limnohabitans lineages. We focused on the LimB lineage of this genus, which is globally distributed and often abundant, and its abundance has shown to be largely invariant to environmental change. Our data show that the LimB lineage is actually comprised of multiple cooccurring populations for which the composition and genomic characteristics are associated with variations in temperature and nutrient levels. The gene expression profiles of this lineage suggest the importance of chemotaxis and motility, traits that had not yet been associated with the Limnohabitans genus, in adapting to environmental conditions.

Bacteria accumulate small, organic compounds called compatible solutes via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. The halophile Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (encoded by ectABC-asp_ect) and glycine betaine (encoded by betIBA-proXWV), four betaine-carnitine-choline transporters (encoded by bccT1 to bccT4), and a second ProU transporter (encoded by proVWX). All of these systems are osmotically inducible with the exception of bccT2. Previously, it was shown that CosR, a MarR-type regulator, was a direct repressor of ectABC-asp_ect in Vibrio species. In this study, we investigated whether CosR has a broader role in the osmotic stress response. Expression analyses demonstrated that betIBA-proXWV, bccT1, bccT3, bccT4, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant showed that expression of these systems is derepressed in the mutant at low salinity compared with the wild type. DNA binding assays demonstrated that purified CosR binds directly to the regulatory region of both biosynthesis systems and four transporters. In Escherichia coli green fluorescent protein (GFP) reporter assays, we demonstrated that CosR directly represses transcription of betIBA-proXWV, bccT3, and proVWX. Similar to Vibrio harveyi, we showed betIBA-proXWV was directly activated by the quorum-sensing LuxR homolog OpaR, suggesting a conserved mechanism of regulation among Vibrio species. Phylogenetic analysis demonstrated that CosR is ancestral to the Vibrionaceae family, and bioinformatics analysis showed widespread distribution among Gammaproteobacteria in general. Incidentally, in Aliivibrio fischeri, Aliivibrio finisterrensis, Aliivibrio sifiae, and Aliivibrio wodanis, an unrelated MarR-type regulator gene named ectR was clustered with ectABC-asp, which suggests the presence of another novel ectoine biosynthesis regulator. Overall, these data show that CosR is a global regulator of osmotic stress response that is widespread among bacteria.

IMPORTANCE Vibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems need to be repressed. However, the mechanism(s) of this repression is not known. In this study, we showed that CosR played a major role in the regulation of multiple compatible solute systems. Phylogenetic analysis showed that CosR is present in all members of the Vibrionaceae family as well as numerous Gammaproteobacteria. Collectively, these data establish CosR as a global regulator of the osmotic stress response that is widespread in bacteria, controlling many more systems than previously demonstrated.

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:

Care services have not been sufficiently adapted to meet the comprehensive care needs of women living with HIV. Our study objective was to engage patients and providers in codesigning care recommendations to improve care for this population in the province of Quebec.

Methods:

We conducted a 5-hour deliberative dialogue workshop in April 2019 in Montréal as the final phase of a mixed-methods study investigating comprehensive care for women living with HIV. The study drew on data from the Canadian HIV Women’s Sexual and Reproductive Health Cohort Study (CHIWOS). Recruitment was guided by a purposive maximum-variation sampling strategy to ensure an appropriate mix of participants and was facilitated by our existing CHIWOS networks. Participants included patients (women living with HIV) and HIV care providers (doctors, nurses, pharmacists). The workshop was facilitated professionally and included a synthesis of the evidence, small- and large-group deliberations, and voting on care improvements.

Results:

Eight patients and 8 HIV care providers participated. Drawing on identified care priorities, the participants identified 4 relatively rapid care improvements and 3 longer-term improvements. The rapid care improvements included delegating medical acts to members of multidisciplinary care teams; greater involvement of HIV community members within care settings and health care decision-making; creating a women’s health information booklet; and increasing HIV education among all health care providers and raising awareness of women’s care needs beyond HIV-specific care among HIV care providers. The longer-term care improvements included advocating for complete financial coverage of antiretroviral therapy within the government-sponsored Medicare program, facilitating access to allied care providers (e.g., physiotherapists and psychologists) and launching a population-wide campaign to increase awareness about the Undetectable = Untransmittable (U=U) initiative and other HIV advances.

Interpretation:

The deliberative dialogue workshop yielded evidence-based, stakeholder-driven recommendations to improve the comprehensive care of women living with HIV in Quebec.

Background:

Patients with multimorbidity often require services across different health care settings, yet team processes among settings are rarely implemented. We explored perceptions of specialists and family physicians collaborating in a telemedicine interprofessional consultation for patients with multimorbidity to better understand the value of bringing physicians together across the boundaries of health care settings.

Methods:

This was a descriptive qualitative, interview-based study. Physicians who had previously participated in the Telemedicine Interprofessional Model of Practice for Aging and Complex Treatments (Telemedicine IMPACT Plus [TIP] Program) were invited to participate and asked to describe their experience of being a member of the program. Interviews were conducted from March to May 2016. We conducted an iterative and interpretive process using both individual and team analysis to identify themes.

Results:

There were 15 participants, 9 specialists and 6 family physicians. Three themes emerged in the analysis: creating new perspectives on care for patients with multimorbidity by sharing knowledge, skills and attitudes; the shift from a consultant model to an interprofessional team model (allowing a window into the community, extending discussions beyond the medical model and focusing on the patient’s health in context); and opportunities for learners, including learning about interprofessional collaboration and gaining exposure to a real-world model for caring for people with multimorbidity in outpatient settings.

Interpretation:

Family physicians and specialists participating in a TIP Program believed the program improved their knowledge and skills, while also serving as an effective care delivery strategy. The findings also support that learners require more exposure to nontraditional consultant models in order to care for patients with multimorbidity effectively.

Background:

Female physicians have been shown to receive fewer awards from medical societies than their male colleagues. We examined the sex distribution of recipients of Canadian residency association awards.

Methods:

We conducted a retrospective observational study of the sex of staff and resident physician recipients of resident-selected awards from provincial and national residency associations using data from 2000–2018. We classified awards into professionalism, advocacy and wellness awards, and education and teaching awards based on award names and descriptions, and compared the proportion of male and female recipients in these categories.

Results:

We identified 314 recipients of staff physician awards and 129 recipients of resident physician awards. Male staff and resident physicians had higher odds of receiving awards than their female counterparts (odds ratio [OR] 1.45, 95% confidence interval [CI] 1.13–1.89 and OR 1.70, 95% CI 1.18–2.46, respectively). There was a reduction in the odds of male residents’ receiving an award over the study period (OR 0.94, 95% CI 0.90–0.98). Male physicians had higher odds of receiving education and teaching awards than female physicians as staff but not as residents (OR 3.21, 95% CI 1.72–5.95 and OR 1.96, 95% CI 0.84–4.60, respectively).

Interpretation:

Male staff and resident physicians in Canada had higher odds of receiving awards from provincial and national residency associations between 2000 and 2018 than their female counterparts. Given this disparity, it would be prudent for organizations that distribute awards to physicians, residents and medical students to examine their nomination criteria and processes for potential bias.

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.

Cushing syndrome results from chronic excessive exposure to glucocorticoids, impacting virtually every organ system with the most dominant effects on fat metabolism, immune function, and musculoskeletal systems. Endogenous Cushing syndrome is rare, most usually due to excess ACTH secretion from pituitary, and less frequently from ectopic tumors. Other cases result from ACTH-independent adrenal overproduction of cortisol.

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This interesting case report from South Africa focuses on a difficult diagnostic challenge: apparent Cushing syndrome with inconsistent laboratory findings.

Cancer continues to be one of the leading causes of morbidity and mortality in the US. Although the treatment of cancer has evolved over the past decades with the use of targeted therapies and immunotherapy, many of these new treatments are expensive and are not readily available to everyone. Moreover, the recent success with treatment advances are not generalized to all cancer types, as some cancers continue to be devastating without significant progress in treatment options. Hence, early detection through population screening remains a critical armament against cancer.

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Genetic variants are the primary driver of congenital heart disease (CHD) pathogenesis. However, our ability to identify causative variants is limited. To identify causal CHD genes that are associated with specific molecular functions, the study used prior knowledge to filter de novo variants from 2,881 probands with sporadic severe CHD. This approach enabled the authors to identify an association between left ventricular outflow tract obstruction lesions and genes associated with the WAVE2 complex and regulation of small GTPase-mediated signal transduction. Using CRISPR zebrafish knockdowns, the study confirmed that WAVE2 complex proteins brk1, nckap1, and wasf2 and the regulators of small GTPase signaling cul3a and racgap1 are critical to cardiac development.

In low-dose computed tomography (LDCT) screening for lung cancer, all three main conditions for overdiagnosis in cancer screening are present: 1) a reservoir of slowly or nongrowing lung cancer exists; 2) LDCT is a high-resolution imaging technology with the potential to identify this reservoir; and 3) eligible screening participants have a high risk of dying from causes other than lung cancer. The degree of overdiagnosis in cancer screening is most validly estimated in high-quality randomised controlled trials (RCTs), with enough follow-up time after the end of screening to avoid lead-time bias and without contamination of the control group.

Nine RCTs investigating LDCT screening were identified. Two RCTs were excluded because lung cancer incidence after the end of screening was not published. Two other RCTs using active comparators were also excluded. Therefore, five RCTs were included: two trials were at low risk of bias, two of some concern and one at high risk of bias. In a meta-analysis of the two low risk of bias RCTs including 8156 healthy current or former smokers, 49% of the screen-detected cancers were overdiagnosed. There is uncertainty about this substantial degree of overdiagnosis due to unexplained heterogeneity and low precision of the summed estimate across the two trials.

Key points

Nine randomised controlled trials (RCTs) on low-dose computed tomography screening were identified; five were included for meta-analysis but only two of those were at low risk of bias.

In a meta-analysis of recent low risk of bias RCTs including 8156 healthy current or former smokers from developed countries, we found that 49% of the screen-detected cancers may be overdiagnosed.

There is uncertainty about the degree of overdiagnosis in lung cancer screening due to unexplained heterogeneity and low precision of the point estimate.

If only high-quality RCTs are included in the meta-analysis, the degree of overdiagnosis is substantial.

Educational aims

To appreciate that low-dose computed tomography screening for lung cancer meets all three main conditions for overdiagnosis in cancer screening: a reservoir of indolent cancers exists in the population; the screening test is able to "tap" this reservoir by detecting biologically indolent cancers as well as biologically important cancers; and the population being screened is characterised by a relatively high competing risk of death from other causes

To learn about biases that might affect the estimates of overdiagnosis in randomised controlled trials in cancer screening

Chronic obstructive pulmonary disease (COPD) is characterised by persistent respiratory symptoms and airflow limitation, which is caused by small airway disease (bronchiolitis) and alveolar destruction (emphysema) [1]. Patients primarily suffering from severe emphysema are often limited in exercise capacity due to the consequences of hyperinflation [2].

Recently, there has been a worldwide resurgence in pneumoconiosis, or pulmonary fibrosis due to occupational mineral dust exposure. In Queensland, Australia, there has been a re-emergence of coal workers' pneumoconiosis and silicosis. Some coal mining communities have experienced a resurgence of progressive massive fibrosis in the USA and a worldwide epidemic is occurring of accelerated silicosis due to exposure to artificial stone.

These diseases are all preventable and should not be occurring in the 21st century. Best practice prevention includes reduction of exposure to mineral dusts or, ideally, prevention of exposure altogether. However, where dust exposure has occurred, respiratory surveillance can provide a strategy for early disease detection. It is important to identify early signs of occupational lung disease at a stage where intervention may be beneficial, though it must be acknowledged that progression may occur even after cessation of exposure to dusts. Respiratory surveillance should be distinguished from population screening and case finding, which are different methods used for disease investigation and control. Designing an ideal respiratory surveillance programme is challenging, as there is no single test that accurately identifies early disease. Several different respiratory disorders may occur related to the same exposure(s). Physicians organising and interpreting tests used in respiratory surveillance must be aware of the broad range of potential work-related respiratory conditions, complexities in diagnosis, and appropriate interpretation of the exposure history, as well as current management options. A working knowledge of the compensation and medicolegal avenues available to workers in individual jurisdictions is also useful.

Key points

Mineral dust exposure causes a number of conditions, including those specific to dust exposures, such as the pneumoconioses (or pulmonary fibroses due to mineral dust exposure), and others that may additionally be related to other causes, such as COPD.

Identification of multiple conditions using respiratory investigations requires expert interpretation and understanding of the range of potential conditions.

The frequency and content of a respiratory surveillance programme will vary according to the relevant occupational exposures, and be affected by both medical and nonmedical factors, including the background prevalence of local diseases. A programme will also need to consider other factors such as local legislation, availability of resources, worker convenience and cost.

Educational aims

To identify the large range of respiratory diseases caused by exposure to mineral dusts and identify the range of tests that may be used in a surveillance programme for occupational respiratory disorders.

To highlight difficulties that might be experienced by medical practitioners in designing and operating an effective surveillance programme, while incorporating rapidly advancing medical technology and practice.

Some years ago, I entered a completely unfamiliar world. This was a landscape that clinicians deal with every day but for the individual suspected of having lung cancer, it can appear hostile and scary, often misrepresented by outdated imagery, information and television portrayal. Lung cancer is not awash with celebrities admitting to having it or grand fundraising campaigns like other conditions. Despite many changes in the treatment landscape, it's still generally much more stigmatised than other cancers.

Since lung cancer (LC) is still the leading cause of cancer deaths worldwide [1], early detection through screening represents an important opportunity to improve LC survival and is a priority area for cancer care. The National Lung Screening Trial (NLST) aimed to compare low-dose helical computed tomography (LDCT) with chest radiography in LC screening of current or former heavy smokers. The trial found a relative reduction in mortality from LC of 20% in those who had undergone LDCT screening. LC screening has regained prominence in the thoracic oncology literature with the completion of NELSON and other European trials, which support the role of LC screening in achieving early diagnosis and reducing mortality. A growing number of implementation pilots are providing an impetus towards organised, national programmes for LC screening, which are in need of long-term follow-up data such as those presented in this study.

Venous thromboembolism (VTE), as a term that encompasses pulmonary embolism (PE) and deep vein thrombosis (DVT), is one of the leading causes of maternal morbidity and mortality [1], especially in developed countries, where PE takes second place after complications of hypertensive disorders [2]. When compared to non-pregnant women of similar age, pregnant women have an approximately four to five times higher risk of VTE [3], with an incidence of 1 in 1000 pregnancies [4]. Approximately 20–25% of VTE cases are caused by PE and 75–80% of cases are caused by DVT [5]. About 60% of DVT occurs antepartum, with the highest risk of antepartum pregnancy-associated VTE being in the third trimester.However, about 60% of PE occurs postpartum [3].

A 78-year-old male presented at the emergency room complaining of dry cough, fever up to 38.5 °C and malaise for 1 month. He had visited a general practitioner and received amoxicillin 500 mg three times a day for 7 days for a presumed chest infection, without improvement. He had a history of diabetes and arterial blood hypertension, for which he was receiving metformin 1000 mg twice a day and amlodipine 10 mg a day for 7 years. He reported no alcohol abuse and was an ex-smoker of 20 pack-years (quit 30 years ago). He had no recent hospitalisations or any medical interventions.

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.

Intrathecal administration of anti-infectives is indicated in central nervous system infections by multiresistant pathogens when drugs that can reach adequate cerebrospinal fluid (CSF) concentrations by systemic therapy are not available. Antibiotics that readily pass the blood-brain and blood-CSF barriers and/or that have low toxicity allowing an increase in the daily dosage should not be used for intrathecal therapy. Intrathecal therapy is accompanied by systemic treatment. Antibacterials indispensable for intrathecal therapy include aminoglycosides, colistin, daptomycin, tigecycline, and vancomycin. Limited experience suggests the utility of the antifungals amphotericin B and caspofungin. Intraventricular administration ensures distribution throughout the CSF compartment, whereas intralumbar dosing often fails to attain adequate antibiotic concentrations in the ventricles. The individual dose is determined by the estimated size of the CSF space and by the estimated clearance from CSF. For moderately lipophilic anti-infectives with a molecular weight above approximately 1,000 g/mol, as well as for hydrophilic drugs with a molecular weight above approximately 400 g/mol, one daily dose is normally adequate. The ventricular drain should be clamped for 15 to 120 min to facilitate the distribution of the anti-infective in the CSF space. Therapeutic drug monitoring of the trough levels is necessary only in cases of therapeutic failure.

Although not as ubiquitous as antibacterial susceptibility testing, antifungal susceptibility testing (AFST) is a tool of increasing importance in clinical microbiology laboratories. The goal of AFST is to reliably produce MIC values that may be used to guide patient therapy, inform epidemiological studies, and track rates of antifungal drug resistance. There are three methods that have been standardized by standards development organizations: broth dilution, disk diffusion, and azole agar screening for Aspergillus. Other commonly used methods include gradient diffusion and the use of rapid automated instruments. Novel methodologies for susceptibility testing are in development. It is important for laboratories to consider not only the method of testing but also the interpretation (or lack thereof) of in vitro data.

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:

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