Antimicrobial stewardship in the primary care setting: from dream to reality?

BACKGROUND: Clinicians who work in primary care are potentially the most influential healthcare professionals to address the problem of antibiotic resistance because this is where most antibiotics are prescribed. Despite a number of evidence based interventions targeting the management of community infections, the inappropriate antibiotic prescribing rates remain high. DISCUSSION: The question is how can appropriate prescribing of antibiotics through the use of Antimicrobial Stewardship (AMS) programs be successfully implemented in primary care. We discuss that a top-down approach utilising a combination of strategies to ensure the sustainable implementation and uptake of AMS interventions in the community is necessary to support clinicians and ensure a robust implementation of AMS in primary care. Specifically, we recommend a national accreditation standard linked to the framework of Core Elements of Outpatient Antibiotic Stewardship, supported by resources to fund the implementation of AMS interventions that are connected to quality improvement initiatives. This article debates how this can be achieved. The paper highlights that in order to support the sustainable uptake of AMS programs in primary care, an approach similar to the hospital and post-acute care settings needs to be adopted, utilising a combination of behavioural and regulatory processes supported by sustainable funding. Without these strategies the problem of inappropriate antibiotic prescribing will not be adequately addressed in the community and the successful implementation and uptake of AMS programs will remain a dream.

Meta-analysis of randomized and quasi-randomized clinical trials of topical antibiotics after primary closure for the prevention of surgical-site infection.

BACKGROUND: Surgical-site infections (SSIs) increase patient morbidity and costs. The aim was to identify and synthesize all RCTs evaluating the effect of topical antibiotics on SSI in wounds healing by primary intention. METHODS: The search included Ovid MEDLINE, Ovid Embase, the Cochrane Wounds Specialized Register, Central Register of Controlled Trials and EBSCO CINAHL from inception to May 2016. There was no restriction of language, date or setting. Two authors independently selected studies, extracted data and assessed risk of bias. When sufficient numbers of comparable trials were available, data were pooled in meta-analysis. RESULTS: Fourteen RCTs with 6466 participants met the inclusion criteria. Pooling of eight trials (5427 participants) showed that topical antibiotics probably reduced the risk of SSI compared with no topical antibiotic (risk ratio (RR) 0·61, 95 per cent c.i. 0·42 to 0·87; moderate-quality evidence), equating to 20 fewer SSIs per 1000 patients treated. Pooling of three trials (3012 participants) for risk of allergic contact dermatitis found no clear difference between antibiotics and no antibiotic (RR 3·94, 0·46 to 34·00; very low-quality evidence). Pooling of five trials (1299 participants) indicated that topical antibiotics probably reduce the risk of SSI compared with topical antiseptics (RR 0·49, 0·30 to 0·80; moderate-quality evidence); 43 fewer SSIs per 1000 patients treated. Pooling of two trials (541 participants) showed no clear difference in the risk of allergic contact dermatitis with antibiotics or antiseptic agents (RR 0·97, 0·52 to 1·82; very low-quality evidence). CONCLUSION: Topical antibiotics probably prevent SSI compared with no topical antibiotic or antiseptic. No conclusion can be drawn regarding whether they cause allergic contact dermatitis.

Triptan use in Australia 1997-2015: A pharmacoepidemiological study.

OBJECTIVE: This study examined the use of triptan derivatives in Australia between 1997 and 2015, based on a national drug reimbursement database, and compared patterns of use with available international data. METHODS: We obtained publically available data on the number of prescriptions for triptans marketed in Australia (sumatriptan, eletriptan, rizatriptan, zolmitriptan, naratriptan). Dispensed use was measured as defined daily dose (DDD per 1000 population per day) for Australia's concessional beneficiaries (low-income earners, people with disabilities, and seniors). RESULTS: Total triptan use increased at an average annual rate of 112% over the 18-year period. Sumatriptan was the preferred triptan throughout (average annual increase 45%). Zolmitriptan and naratriptan use peaked in 2004, then decreased. Rizatriptan and eletriptan became available in 2010. There were 3.2-fold and 5.9-fold annual increases in their use from 2011 to 2105. There was some evidence suggesting that pattern of triptan use in concessional beneficiaries probably reflected pattern of overall triptan use in Australia. CONCLUSIONS: The use of triptan derivatives in Australia per head of population for treating migraine attacks continued to increase over the 18-year period studied, with use of recently introduced derivatives more than substituting for decreased use of older triptans. This suggests that the available treatments of migraine attacks had achieved what were considered less than adequate therapeutic outcomes.

Off-label use of tumour necrosis factor-alpha inhibitors and anakinra at an Australian tertiary hospital.

BACKGROUND: Tumour necrosis factor-alpha inhibitors (anti-TNFα) and anakinra are monoclonal antibodies against pro-inflammatory cytokines overexpressed in many systemic inflammatory diseases. In Australia, they are registered for the treatment of several rheumatological, gastroenterological and dermatological indications. Despite increasing observational evidence for their use in off-label indications, there is a paucity of outcome research from the Australian hospital sector. AIMS: To describe the off-label use of anti-TNFα and anakinra at a tertiary referral hospital in Queensland, Australia and consideration of a drug register to inform future clinical decision-making. METHODS: We performed an in-depth retrospective chart audit of off-label treatment with anti-TNFα or anakinra at the Royal Brisbane and Women's Hospital from mid-2010 to mid-2014, linking demographic, phenotypic, pathology and outcome data with these drugs. RESULTS: Off-label use was identified in 10 patients. The most frequent indications were sarcoidosis and dermatological conditions. Three patients required sequential therapy with a second anti-TNFα (total responses = 13). Complete response occurred in 46%, partial response in 38% and primary non-response in 8%. Response was unable to be determined in 8%. We recorded 14 adverse events (infections most common). CONCLUSION: This study suggests that anti-TNFα may be beneficial for some off-label indications (e.g. sarcoidosis). However, the observational design of this study (and pre-existing research) limits the ability to infer causality and generalise results. We propose the creation of a mandatory drug register to monitor off-label use. Whilst comparative efficacy cannot be established without a matched placebo arm, a register would enable some reporting on effectiveness in rare diseases and identify infrequent but serious adverse events.

Anticholinergic medicines in an older primary care population: a cross-sectional analysis of medicines' levels of anticholinergic activity and clinical indications.

WHAT IS KNOWN AND OBJECTIVES: Adverse clinical outcomes have been associated with cumulative anticholinergic burden (to which low-potency as well as high-potency anticholinergic medicines contribute). The clinical indications for which anticholinergic medicines are prescribed (and thus the 'phenotype' of patients with anticholinergic burden) have not been established. We sought to establish the overall prevalence of prescribing of anticholinergic medicines, the prevalence of prescribing of low-, medium- and high-potency anticholinergic medicines, and the clinical indications for which the medicines were prescribed in an older primary care population. METHODS: This was a cross-sectional analysis of a cohort study of Australian early-career general practitioners' (GPs') clinical consultations - the Registrar Clinical Encounters in Training (ReCEnT) study. In ReCEnT, GPs collect detailed data (including medicines prescribed and their clinical indication) for 60 consecutive patients, on up to three occasions 6 months apart. Anticholinergic medicines were categorized as levels 1 (low-potency) to 3 (high-potency) using the Anticholinergic Drug Scale (ADS). RESULTS: During 2010-2014, 879 early-career GPs (across five of Australia's six states) conducted 20 555 consultations with patients aged 65 years or older, representing 35 506 problems/diagnoses. Anticholinergic medicines were prescribed in 10·4% [95% CIs 9·5-10·5] of consultations. Of the total anticholinergic load of prescribed medicines ('community anticholinergic load') 72·7% [95% CIs 71·0-74·3] was contributed by Level 1 medicines, 0·8% [95% CIs 0·5-1·3] by Level 2 medicines and 26·5% [95% CIs 24·8-28·1] by Level 3 medicines. Cardiac (40·0%), Musculoskeletal (16·9%) and Respiratory (10·6%) were the most common indications associated with Level 1 anticholinergic prescription. For Level 2 and 3 medicines (combined data), Psychological (16·1%), Neurological (16·1%), Musculoskeletal (15·7%) and Urological (11·1%) indications were most common. WHAT IS NEW AND CONCLUSION: Anticholinergic medicines are frequently prescribed in Australian general practice, and the majority of the 'community' anticholinergic burden is contributed by 'low'-anticholinergic potency medicines whose anticholinergic effects may be largely 'invisible' to prescribing GPs. Furthermore, the clinical 'phenotype' of the patient with high anticholinergic burden may be very different to common stereotypes (patients with urological, psychological or neurological problems), potentially making recognition of risk of anticholinergic adverse effects additionally problematic for GPs.

Management of Postthyroidectomy Hypoparathyroidism and Its Effect on Hypocalcemia-Related Complications: A Meta-Analysis.

OBJECTIVE: The aim of this Meta-analysis is to evaluate the impact of different treatment strategies for early postoperative hypoparathyroidism on hypocalcemia-related complications and long-term hypoparathyroidism. DATA SOURCES: Embase.com, MEDLINE, Web of Science Core Collection, Cochrane Central Register of Controlled Trials, and the top 100 references of Google Scholar were searched to September 20, 2022. REVIEW METHODS: Articles reporting on adult patients who underwent total thyroidectomy which specified a treatment strategy for postthyroidectomy hypoparathyroidism were included. Random effect models were applied to obtain pooled proportions and 95% confidence intervals. Primary outcome was the occurrence of major hypocalcemia-related complications. Secondary outcome was long-term hypoparathyroidism. RESULTS: Sixty-six studies comprising 67 treatment protocols and 51,096 patients were included in this Meta-analysis. In 8 protocols (3806 patients), routine calcium and/or active vitamin D medication was given to all patients directly after thyroidectomy. In 49 protocols (44,012 patients), calcium and/or active vitamin D medication was only given to patients with biochemically proven postthyroidectomy hypoparathyroidism. In 10 protocols (3278 patients), calcium and/or active vitamin D supplementation was only initiated in case of clinical symptoms of hypocalcemia. No patient had a major complication due to postoperative hypocalcemia. The pooled proportion of long-term hypoparathyroidism was 2.4% (95% confidence interval, 1.9-3.0). There was no significant difference in the incidence of long-term hypoparathyroidism between the 3 supplementation groups. CONCLUSIONS: All treatment strategies for postoperative hypocalcemia prevent major complications of hypocalcemia. The early postoperative treatment protocol for postthyroidectomy hypoparathyroidism does not seem to influence recovery of parathyroid function in the long term.

Mutations in a human homologue of Drosophila crumbs cause retinitis pigmentosa (RP12).

Retinitis pigmentosa (RP) comprises a clinically and genetically heterogeneous group of diseases that afflicts approximately 1.5 million people worldwide. Affected individuals suffer from a progressive degeneration of the photoreceptors, eventually resulting in severe visual impairment. To isolate candidate genes for chorioretinal diseases, we cloned cDNAs specifically or preferentially expressed in the human retina and the retinal pigment epithelium (RPE) through a novel suppression subtractive hybridization (SSH) method. One of these cDNAs (RET3C11) mapped to chromosome 1q31-q32.1, a region harbouring a gene involved in a severe form of autosomal recessive RP characterized by a typical preservation of the para-arteriolar RPE (RP12; ref. 3). The full-length cDNA encodes an extracellular protein with 19 EGF-like domains, 3 laminin A G-like domains and a C-type lectin domain. This protein is homologous to the Drosophila melanogaster protein crumbs (CRB), and denoted CRB1 (crumbs homologue 1). In ten unrelated RP patients with preserved para-arteriolar RPE, we identified a homozygous AluY insertion disrupting the ORF, five homozygous missense mutations and four compound heterozygous mutations in CRB1. The similarity to CRB suggests a role for CRB1 in cell-cell interaction and possibly in the maintenance of cell polarity in the retina. The distinct RPE abnormalities observed in RP12 patients suggest that CRB1 mutations trigger a novel mechanism of photoreceptor degeneration.

An innovative antimicrobial stewardship programme for children in remote and regional areas in Queensland, Australia: optimising antibiotic use through timely intravenous-to-oral switch.

OBJECTIVES: Little is known about the benefits of timely switch from intravenous (IV) to oral antibiotic therapy in children. We evaluated the appropriateness of IV-to-oral switch of antibiotic therapy in remote and regional areas of Australia following the implementation of a multifaceted package of interventions. METHODS: The intervention package, including clinician guidelines, medication review stickers, patient information leaflets and educational resources, was implemented in seven facilities in Queensland, Australia. Children with community-acquired pneumonia and skin and soft-tissue infections were switched to oral therapy if they met the required 'IV-to-oral switch' criteria. Data were collected for a 7-month period from May to November for the baseline (2018) and intervention (2019) phases. RESULTS: A total of 357 patients were enrolled in the study, including 178 in the baseline phase and 179 in the intervention phase. The percentage of patients who switched to oral therapy or stopped IV antibiotics, within 24 h of eligibility, increased from 87.6% (156/178) in the baseline phase to 97.2% (174/179) in the intervention phase (P = 0.003). The average number of extra IV days decreased from 0.45 days in the baseline period to 0.18 days in the intervention period (P < 0.001). The median patient length of stay was 2 days for both phases. The only adverse events recorded were line-associated infiltration, with a decrease from 34.3% (61/178) (baseline) to 17.9% (32/179) (intervention) (P < 0.001). CONCLUSION: A multifaceted intervention package to enhance timely IV-to-oral switch of antibiotic therapy for children in remote and regional facilities is effective.

Assessment of immune function after short-term administration of recombinant human growth hormone in healthy young males.

Growth hormone (GH) is a commonly used drug aimed at improving sport performance. The aim of this study is to evaluate the immunomodulatory effects of short-term administration of recombinant GH (rhGH) in healthy young males. NK cell number, activity and phenotype, T cell number, CD4(+) (Th1/Th2) cytokine production of IL2, IL4, IL6, IL10, TNF-α and IFN-γ and CD4(+)/CD8(+) ratio with particular attention to the possible correlation to IGF-I production were investigated. 30 males (27 ± 9 years) were randomly assigned to placebo (n = 15) or drug (rhGH) 1 mg/day groups (n = 15) with daily injection for 7 days. IGF-I plasma concentration and flow cytometry data were generated at baseline and days 8, 15, 22 and 29 post injection. Data analysis used General Linear Model with repeated measures, Bonferroni correction factor and significance at p ≤ 0.05. Serum IGF-I levels (ng/mL) increased significantly (p ≤ 0.01) on day 8 (0.48 ± 0.78) after injections compared to baseline (0.31 ± 0.07) and days 15 (0.33 ± 0.06), 22 (0.29 ± 0.05) and 29 (0.29 ± 0.06). A significant time effect was noted in IL10 secretion (pg/mL) from day 15 (P = 35.14 ± 19.93, rhGH = 26.63 ± 16.39) to days 22 (P = 61.32 ± 20.41, rhGH = 74.99 ± 46.91) and 29 (P = 101.98 ± 67.25, rhGH = 107.74; ± 122.58). There was no correlation between IGF-I and NK activity, phenotype or number along with T lymphocyte number, CD4(+)/CD8(+) ratio or Th1 and Th2 cytokine production. In conclusion, cytokine secretion spectrum was not affected by short-term rhGH administration in young males.

Trends in Australian dental prescribing of antibiotics: 2005-2016.

BACKGROUND: Prescribing of antibiotics by dentists for surgical prophylaxis or as an adjunct to managing dental infections is a substantial part of the overall landscape for prescribed antibiotics in health care settings. METHODS: We explored trends in the antibiotic prescribing patterns of Australian dentists over the 12-year period, 2005-2016. We obtained data on dispensed prescriptions of antibiotics from registered dentists subsidized on the Pharmaceutical Benefits Scheme. RESULTS: Australian dentists were responsible for almost 7 million dispensed prescriptions of antibiotics over 12 years; an average of 24 prescriptions per year per dentist. The most commonly prescribed antibiotic was amoxicillin, followed by amoxicillin + clavulanic acid and metronidazole. These top three antibiotics constituted more than 80% of all antibiotics prescribed and their use increased dramatically over time. There was a large increase in the prescribing of broad-spectrum antibiotics over time, most of which occurred from 2011 to 2016. CONCLUSIONS: Excessive prescribing of broad-spectrum antibiotics runs contrary to national antimicrobial stewardship (AMS) initiatives and guidelines. Multifaceted educational strategies are essential to align prescribing with current best practice. High-level evidence to inform clear guidelines on antibiotic prescribing in dental infections, with audit and feedback, should reduce the inappropriate use of antibiotics in dentistry.

Visual Analytics for Hypothesis-Driven Exploration in Computational Pathology.

Recent advances in computational and algorithmic power are evolving the field of medical imaging rapidly. In cancer research, many new directions are sought to characterize patients with additional imaging features derived from radiology and pathology images. The emerging field of Computational Pathology targets the high-throughput extraction and analysis of the spatial distribution of cells from digital histopathology images. The associated morphological and architectural features allow researchers to quantify and characterize new imaging biomarkers for cancer diagnosis, prognosis, and treatment decisions. However, while the image feature space grows, exploration and analysis become more difficult and ineffective. There is a need for dedicated interfaces for interactive data manipulation and visual analysis of computational pathology and clinical data. For this purpose, we present IIComPath, a visual analytics approach that enables clinical researchers to formulate hypotheses and create computational pathology pipelines involving cohort construction, spatial analysis of image-derived features, and cohort analysis. We demonstrate our approach through use cases that investigate the prognostic value of current diagnostic features and new computational pathology biomarkers.

18-year experience in the management of men with a complaint of a small penis.

In many cultures, the erect penis has been a symbol of masculine qualities. Because of this symbolism, a penis that is less than average size can cause insecurity or embarrassment. This series reports the authors' 18-year experience in the management of 60 men with a complaint of a small penis. For 44 of these 60 men, counseling was sufficient; the other 16 had surgery, and of these, 9 were satisfied with the result. Despite limitations, the authors conclude that those men who already achieve a penis length of no less than 7.5 cm (2.95 in) in erection, have only limited benefit from penis-enhancing surgery. This particular patient category should therefore be dissuaded from surgery.

Penile enlargement: from medication to surgery.

Penis lengthening pills, stretch apparatus, vacuum pumps, silicone injections, and lengthening and thickening operations are available for men who worry about their penis size. Surgery is thus far the only proven scientific method for penile enlargement. In this article, we consider patient selection, outcome evaluation, and techniques applied. In our view, sexological counseling and detailed explanation of risks and complications are mandatory before any operative intervention.

Thyroid hormones treatment for subclinical hypothyroidism: a clinical practice guideline.

CLINICAL QUESTION: What are the benefits and harms of thyroid hormones for adults with subclinical hypothyroidism (SCH)? This guideline was triggered by a recent systematic review of randomised controlled trials, which could alter practice. CURRENT PRACTICE: Current guidelines tend to recommend thyroid hormones for adults with thyroid stimulating hormone (TSH) levels >10 mIU/L and for people with lower TSH values who are young, symptomatic, or have specific indications for prescribing. RECOMMENDATION: The guideline panel issues a strong recommendation against thyroid hormones in adults with SCH (elevated TSH levels and normal free T4 (thyroxine) levels). It does not apply to women who are trying to become pregnant or patients with TSH >20 mIU/L. It may not apply to patients with severe symptoms or young adults (such as those ≤30 years old). HOW THIS GUIDELINE WAS CREATED: A guideline panel including patients, clinicians, and methodologists produced this recommendation in adherence with standards for trustworthy guidelines using the GRADE approach. THE EVIDENCE: The systematic review included 21 trials with 2192 participants. For adults with SCH, thyroid hormones consistently demonstrate no clinically relevant benefits for quality of life or thyroid related symptoms, including depressive symptoms, fatigue, and body mass index (moderate to high quality evidence). Thyroid hormones may have little or no effect on cardiovascular events or mortality (low quality evidence), but harms were measured in only one trial with few events at two years' follow-up. UNDERSTANDING THE RECOMMENDATION: The panel concluded that almost all adults with SCH would not benefit from treatment with thyroid hormones. Other factors in the strong recommendation include the burden of lifelong management and uncertainty on potential harms. Instead, clinicians should monitor the progression or resolution of the thyroid dysfunction in these adults. Recommendations are made actionable for clinicians and their patients through visual overviews. These provide the relative and absolute benefits and harms of thyroid hormones in multilayered evidence summaries and decision aids available in MAGIC (https://app.magicapp.org/) to support shared decisions and adaptation of this guideline.

[Priapism following off-label use of tamulosin]. / Priapisme na 'off-label'-gebruik van tamsulosine.

Selective postsynaptic alpha1-adrenergic blocking agents such as tamsulosin are mainly used for treating micturition symptoms due to prostatic hyperplasia. They are also used off-label in patients with a distal ureteral stone to enhance passage of the stone. This application is based on the smooth muscle relaxant effect of alpha1-receptor blockade. Smooth muscle relaxation in the penis leads to penile tumescence. The case was reported of a 47-year-old man with a distal ureteral stone who presented with priapism after using tamsulosin 0.4 mg once daily for 9 days. Male patients receiving off-label alpha1-blocking agents should be informed about this adverse effect.

A novel antiapoptotic mechanism based on interference of Fas signaling by CD44 variant isoforms.

There is growing evidence that one of the central common characteristics of tumor and inflammatory cells is their resistance to programmed cell death. This feature results in the accumulation of harmful cells, which are mostly refractory to Fas (FAS, APO-1)-mediated apoptosis. A molecule found on these cells is the transmembrane receptor CD44 with its variant isoforms (CD44v). The establishment of transfectants expressing different CD44v isoforms allowed us to demonstrate that the CD44v6 and CD44v9 isoforms exhibit an antiapoptotic effect and can block Fas-mediated apoptosis. Moreover, we observed that CD44v6 and CD44v9 colocalize and interact with Fas. Importantly, an anti-CD44v6 antibody can abolish the antiapoptotic effect of CD44v6. These results are the first to show that CD44v isoforms interfere with Fas signaling. Our findings improve the understanding of the pathogenesis of cancer and autoimmunity and open new strategies to treat such disorders.

Evidence for auto/paracrine actions of vitamin D in bone: 1alpha-hydroxylase expression and activity in human bone cells.

Vitamin D is an important regulator of mineral homeostasis and bone metabolism. 1Alpha-hydroxylation of 25-(OH)D3 to form the bioactive vitamin D hormone, 1alpha,25-(OH)2D3, is classically considered to take place in the kidney. However, 1alpha-hydroxylase has been reported at extrarenal sites. Whether bone is a 1alpha,25-(OH)2D3 synthesizing tissue is not univocal. The aim of this study was to investigate an autocrine/paracrine function for 1alpha,25-(OH)2D3 in bone. We show that 1alpha-hydroxylase is expressed in human osteoblasts, as well as the vitamin D binding protein receptors megalin and cubilin. Functional analyses demonstrate that after incubation with the 1alpha-hydroxylase substrate 25-(OH)D3, the osteoblasts can produce sufficient 1alpha,25-(OH)2D3 to modulate osteoblast activity, resulting in induced alkaline phosphatase (ALP) activity, osteocalcin (OC) and CYP24 mRNA expression, and mineralization. The classical renal regulators of 1alpha-hydroxylase, parathyroid hormone, and ambient calcium do not regulate 1alpha-hydroxylase in osteoblasts. In contrast, interleukin (IL)-1beta strongly induces 1alpha-hydroxylase. Besides the bone-forming cells, we demonstrate 1alpha-hydroxylase activity in the bone resorbing cells, the osteoclasts. This is strongly dependent on osteoclast inducer RANKL. This study showing expression, activity, and functionality of 1alpha-hydroxylase unequivocally demonstrates that vitamin D can act in an auto/paracrine manner in bone.

GeneSeeker: extraction and integration of human disease-related information from web-based genetic databases.

The identification of genes underlying human genetic disorders requires the combination of data related to cytogenetic localization, phenotypes and expression patterns, to generate a list of candidate genes. In the field of human genetics, it is normal to perform this combination analysis by hand. We report on GeneSeeker (http://www.cmbi.ru.nl/GeneSeeker/), a web server that gathers and combines data from a series of databases. All database searches are performed via the web interfaces provided with the original databases, guaranteeing that the most recent data are queried, and obviating data warehousing. GeneSeeker makes the same selection of candidate genes as the human geneticists would have performed, and thus reducing the time-consuming process to a few minutes. GeneSeeker is particularly well suited for syndromes in which the disease gene displays altered expression patterns in the affected tissue(s).

Osteoclastogenic capacity of peripheral blood mononuclear cells is not different between women with and without osteoporosis.

INTRODUCTION: Peripheral Blood Mononuclear Cells (PBMCs) have been extensively used as a culture model to generate osteoclasts in vitro. The aim of this study was to assess the osteoclastogenic potential of PBMCs derived from post-menopausal women with longstanding osteoporosis and compare this with PBMCs from healthy controls. MATERIAL AND METHODS: We selected from the population-based Rotterdam Study 82 participants of which 43 were diagnosed with osteoporosis (T-score below -2.5 at the lumbar spine) and the presence of at least 1 fracture and 29 healthy controls (T-score above 1; no fracture). PBMCs were differentiated into osteoclasts, and both differentiation capacity and activity were measured. Total RNA was obtained to assess gene expression of osteoclast markers. Deoxypyridinoline (DPD) was measured in plasma as a marker for bone resorption, in vivo. RESULTS: Neither the number of osteoclasts nor cathepsin K (CTSK) and dendritic cell-specific transmembrane protein (TM7SF4) gene expression was significantly different between both groups. There was also no significant difference in resorption pit area and plasma DPD levels. Stratification by fracture type into a group with vertebral, non-vertebral and both vertebral and non-vertebral fractures showed no difference in osteoclast formation or osteoclastic bone resorption. However, plasma DPD, but not the RNA expression markers, was significantly lower in the group of subjects with vertebral fracture group and those with vertebral and non-vertebral fractures compared to the healthy controls. No differences in osteoclastogenesis, osteoclastic resorption and plasma DPD levels were detected also after exclusion of past or present users of bisphosphonates and glucocorticoids. Stratification into high and low DPD levels showed higher osteoclastogenesis and more osteoclastic bone resorption in the high DPD group compared to the low DPD levels within the group of osteoporotic subjects. CONCLUSION: This study showed no difference in PBMC osteoclastogenic capacity and activity between women with and without osteoporosis and at least one previous fracture, who were on average 29.5years after menopause, suggesting that there is no difference in circulating osteoclast precursors. Although we cannot exclude that circulating precursors may behave differently at the bone site, it is possible that long after menopause a more stable phase of bone turnover is reached compared to earlier after the start of menopause in which differences in circulating osteoclast precursors and osteoclastogenic potential are more prominent.

Complete sequence-based pathway analysis by differential on-chip DNA and RNA extraction from a single cell.

We demonstrate on-chip, differential DNA and RNA extraction from a single cell using a microfluidic chip and a two-stage lysis protocol. This method enables direct use of the whole extract, without additional washing steps, reducing sample loss. Using this method, the tumor driving pathway in individual cells from a colorectal cancer cell line was determined by applying a Bayesian computational pathway model to sequences obtained from the RNA fraction of a single cell and, the mutations driving the pathway were determined by analyzing sequences obtained from the DNA fraction of the same single cell. This combined functional and mutational pathway assessment of a single cell could be of significant value for dissecting cellular heterogeneity in tumors and analyzing single circulating tumor cells.