CounterPath (United States)

Newborn screening is an important public health measure of secondary prevention. With the increasing number of target conditions, there is a growing demand to optimize laboratory processes to save patient material and to work cost-effectively. Here, we report an adaption of the commercially available SpotOn Clinical Diagnostics tandem mass spectrometry test kit to detect hemoglobin fragments as well as substrate-product pairs of biotinidase and porphobilinogen synthase at once. The presence of specific peptides and the enzyme activities allows to infer to disease states, i.e., sickle cell disease, biotinidase deficiency and hereditary tyrosinemia type 1.

Introduction: Nasal carriage of Staphylococcus species plays an important role in the epidemiology and pathogenesis of both community and healthcare-associated infections. Coinciding the emergence of methicillin-resistant Staphylococcus aureus (MRSA) is a challenge for clinicians to prevent their spread. Mupirocin is a topical antimicrobial agent approved for eradicating nasal carriage of staphylococcal species in adult patients and healthcare workers (HCWs). The increasing prevalence of mupirocin resistance among Staphylococcus aureus and coagulase-negative staphylococci species could be an important threat to the future use of mupirocin against MRSA.Objective: The aim of this study is to determine the prevalence of MRSA from nasal swabs of HCWs in intensive care units and its level of resistance pattern of mupirocin in all isolates of Staphylococcus species by disk diffusion and epsilometer test (E-test) and to determine post decolonization screening.Materials and methods: A total of 67 HCWs (doctors, nursing staff, technicians, and housekeeping staff) in the medical and surgical intensive care units were included in the study. Nasal swabs were collected from the subjects and cultured onto nutrient and blood agar, which were then incubated at 37ºC for 18 to 24 hours. Staphylococcus aureus and coagulase-negativeStaphylococcus species (CoNS) were identified by standard biochemical techniques. Methicillin resistance was detected by the disk diffusion method using a 30 µg cefoxitin disk as per the Clinical and Laboratory Standards Institute (CLSI) guidelines, and mupirocin resistance was detected using a 5 µg mupirocin disk. The resistance strains were further subjected to E-strip testing to determine the level of mupirocin resistance.Results: A total of 72 isolates were grown from the 67 subjects used in this study. Nine strains (12.5%) grew S. aureus, and 52 strains (72.2%) grew CoNS. Methicillin resistance was seen in five isolates (6.9%) of S. aureus and 45 isolates (62.5%) of CoNS. Mupirocin resistance was seen in 11 isolates of methicillin-resistant coagulase-negative Staphylococcus species (MRCoNS), where three isolates (4.1%) showed low-level mupirocin resistance MuL and eight isolates (11.11%) showed high-level mupirocin resistance MuH. None of the isolates of MRSA, methicillin-sensitive Staphylococcus aureus (MSSA), and methicillin-sensitive coagulase-negative Staphylococcusspecies (MSCoNS) were resistant to mupirocin. Seven out of nine HCWs (77.8%) showed clearance of the organism after decolonization therapy.Conclusion: The prevalence of emerging resistance to mupirocin in MRSA and MRCoNS is of great concern, especially in the nasal carrier state of HCWs. Hence, methicillin and mupirocin resistance in S. aureus and CoNS must be detected in HCWs as a routine protocol, and decolonization measures should be undertaken to prevent healthcare-associated infections.

The publication and implementation of the 'Finch Group Report' (Accessibility, sustainability, excellence: how to expand access to research publications) in June 2012 is providing a current focus for many of us, especially those based in the UK.Therefore, we are delighted to have articles from Stevan Harnad and Stephen Curry providing their perspectives on the impact of the report.There are some complex points of view in these two articles, and in the articles by Martin Hall and Steven Hall which we published in the November 2012 issue.In our 'Key Issue', Albert Prior has done a fantastic job in pulling together a summary of some of the main views and comments presented by the authors of these four thought-provoking articles.To ensure that we are not presenting a wholly UK-centric view, we are pleased that Reggie Raju and his colleagues from Stellenbosch University have submitted an article reminding us that open access can look very different depending on where in the world you are; and that funding for research in Africa is meagre compared to other parts of the world, meaning that article processing charges (APCs) exclude the majority of Africans from publishing.Nol Verhagen from the University of Amsterdam also provides us with his view on the hybrid open access journal.Our other author from The Netherlands, Ronald Snijder, reminds us that Amsterdam University

Purpose: Pancreatic cysts are frequently detected via imaging, and may have varying potential for malignancy, though most are benign. First line testing (imaging, cytology, and CEA) does not adequately detect malignancy, often resulting in excessive use surgery to manage these cysts. Mutational profiling of pancreatic cysts has emerged as a second-line testing alternative to aid in the determination of the risk of malignancy. We sought to determine the outcomes of patients who had previously been tested with integrated mutational profiling (PathFinderTG, RedPath Integrated Pathology, Pittsburgh, PA) in order to evaluate its performance characteristics. Methods: 362 patients with pancreatic cysts were tested using mutational profiling of pancreatic cyst fluid. Outcomes on 80 patients were determined from review of patient charts. These 80 patients ranged in age from 38 to 94 years (mean 69.6 years, 69% over 65 years). There were 60 females (75%) and 20 males (25%). Malignant diagnoses were confirmed by surgical pathology, positive cytology, or clinical cancer treatment. Benign outcomes were defined as surgical pathology with no malignancy or 2 years with no disease progression. In the present analysis, patients with less than 2 years followup were excluded. For patients who were tested serially over the followup period, the diagnosis from the first case was used as the reference. DNA for mutational profiling was extracted from pancreatic cyst fluid and tested for mutations in a panel of 16 microsatellites targeting 1p, 3p, 5q, 9p, 10q, 17p, 17q, 18q, 21q, 22q plus point mutation in KRAS. Diagnosis incorporated DNA quantity and quality, and integrated relevant imaging or cytologic findings. Overall diagnosis of biological behavior was categorized as Benign, statistically indolent low (SI-Low), statistically indolent high (SI-High), or Aggressive. Diagnoses of Benign and SI-Low were treated as negative for malignancy, and SI-High and Aggressive as positive. Results:Table 1 shows integrated mutational profiling results versus outcome for 80 cyst fluids. Sensitivity and specificity in cyst fluids were 90% and 97%, with accuracy of 96%. The relative risk (RR) of malignancy was 56.5 times greater among cysts diagnosed as positive than as negative.Table 1: No Caption available.Conclusion: Integrated molecular testing of pancreatic cyst fluid shows high overall accuracy and can be a useful addition to first-line testing to assess malignant potential of pancreatic cysts. Disclosure: Damien Mallat - No financial relationship with RedPath Eric Ellsworth - Employee of RedPath Integrated Pathology. Ann-Marie Terry - Employee of RedPath Integrated Pathology. Brendan Corcoran - Employee of RedPath Integrated Pathology Sydney. Finkelstein - Employee, Stockholder, Patent Holder of RedPath Integrated Pathology. This research was supported by an industry grant from RedPath Integrated Pathology paid costs for IRB submission, and paid staff time for retrieval of records. Dr. Mallat received no grants or payment for this study.

Connective tissue growth factor (CTGF) expression is elevated in advanced breast cancer and promotes metastasis. Chemotherapy response is only transient in most metastatic diseases. In the present study, we examined whether CTGF expression could confer drug resistance in human breast cancer. In breast cancer patients who received neoadjuvant chemotherapy, CTGF expression was inversely associated with chemotherapy response. Overexpression of CTGF in MCF7 cells (MCF7/CTGF) enhanced clonogenic ability, cell viability, and resistance to apoptosis on exposure to doxorubicin and paclitaxel. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) mitigated this drug resistance capacity. CTGF overexpression resulted in resistance to doxorubicin- and paclitaxel-induced apoptosis by up-regulation of Bcl-xL and cellular inhibitor of apoptosis protein 1 (cIAP1). Knockdown of Bcl-xL or cIAP1 with specific small interfering RNAs abolished the CTGF-mediated resistance to apoptosis induced by the chemotherapeutic agents in MCF7/CTGF cells. Inhibition of extracellular signal-regulated kinase (ERK)-1/2 effectively reversed the resistance to apoptosis as well as the up-regulation of Bcl-xL and cIAP1 in MCF7/CTGF cells. A neutralizing antibody against integrin alpha(v)beta(3) significantly attenuated CTGF-mediated ERK1/2 activation and up-regulation of Bcl-xL and cIAP1, indicating that the integrin alpha(v)beta(3)/ERK1/2 signaling pathway is essential for CTGF functions. The Bcl-xL level also correlated with the CTGF level in breast cancer patients. We also found that a COOH-terminal domain peptide from CTGF could exert activities similar to full-length CTGF, in activation of ERK1/2, up-regulation of Bcl-xL/cIAP1, and resistance to apoptosis. We conclude that CTGF expression could confer resistance to chemotherapeutic agents through augmenting a survival pathway through ERK1/2-dependent Bcl-xL/cIAP1 up-regulation.

The retinoblastoma (RB) tumor suppressor pathway is likely important in primitive neuroectodermal tumors (PNET) of the brain. In fact, 10% to 15% of children born with RB mutations develop brain PNETs, commonly in the pineal gland. Cyclin D1, which in association with cyclin-dependent kinase (Cdk) 4 and Cdk6 phosphorylates and inactivates the RB protein, is expressed in 40% of sporadic medulloblastoma, a PNET of the cerebellum. To understand tumorigenic events cooperating with RB pathway disruption in brain PNET, we generated a transgenic mouse where cyclin D1 was expressed in pineal cells. Cyclin D1 enhanced pinealocyte proliferation, causing pineal gland enlargement. However, proliferation ceased beyond 2 weeks of age with reversal of Cdk4-mediated Rb phosphorylation despite continued expression of the transgene, and the pineal cells showed heterochromatin foci suggestive of a senescent-like state. In the absence of the p53 tumor suppressor, cell proliferation continued, resulting in pineal PNET that limited mouse survival to approximately 4 months. Interestingly, the Cdk inhibitor p18(Ink4c) was induced in the transgenic pineal glands independently of p53, and transgenic mice that lacked Ink4c developed invasive PNET, although at an older age than those lacking p53. Analogous to our mouse model, we found that children with heritable RB often had asymptomatic pineal gland enlargement that only rarely progressed to PNET. Our finding that the Cdk4 inhibitor p18(Ink4c) is a tumor suppressor in cyclin D1-driven PNET suggests that pharmacologic interventions to inhibit Cdk4 activity may be a useful chemoprevention or therapeutic strategy in cancer driven by primary RB pathway disruption.

Elevated cellular proliferation and cell cycle abnormalities, which have been associated with premalignant lesions, may be caused by inactivation of tumor suppressor genes. We measured proliferative and cell cycle fractions of biopsies from a cohort of patients with Barrett's esophagus to better understand the role of proliferation in early neoplastic progression and the association between cell cycle dysregulation and tumor suppressor gene inactivation. Cell proliferative fractions (determined by Ki67/DNA multiparameter flow cytometry) and cell cycle fractions (DNA content flow cytometry) were measured in 853 diploid biopsies from 362 patients with Barrett's esophagus. The inactivation status of CDKN2A and TP53 was assessed in a subset of these biopsies in a cross-sectional study. A prospective study followed 276 of the patients without detectable aneuploidy for an average of 6.3 years with esophageal adenocarcinoma as an end point. Diploid S and 4N (G(2)/tetraploid) fractions were significantly higher in biopsies with TP53 mutation and loss of heterozygosity. CDKN2A inactivation was not associated with higher Ki67-positive, diploid S, G(1), or 4N fractions. High Ki67-positive and G(1)-phase fractions were not associated with the future development of esophageal adenocarcinoma (P=0.13 and P=0.15, respectively), whereas high diploid S-phase and 4N fractions were (P=0.03 and P<0.0001, respectively). High Ki67-positive proliferative fractions were not associated with inactivation of CDKN2A and TP53 or future development of cancer in our cohort of patients with Barrett's esophagus. Biallelic inactivation of TP53 was associated with elevated 4N fractions, which have been associated with the future development of esophageal adenocarcinoma.