High Resolution Genotyping of Clinical Aspergillus flavus Isolates from India Using Microsatellites

Contains fulltext : 124312.pdf (publisher's version ) (Open Access)BACKGROUND: Worldwide, Aspergillus flavus is the second leading cause of allergic, invasive and colonizing fungal diseases in humans. However, it is the most common species causing fungal rhinosinusitis and eye infections in tropical countries. Despite the growing challenges due to A. flavus, the molecular epidemiology of this fungus has not been well studied. We evaluated the use of microsatellites for high resolution genotyping of A. flavus from India and a possible connection between clinical presentation and genotype of the involved isolate. METHODOLOGY/PRINCIPAL FINDINGS: A panel of nine microsatellite markers were selected from the genome of A. flavus NRRL 3357. These markers were used to type 162 clinical isolates of A. flavus. All nine markers proved to be polymorphic displaying up to 33 alleles per marker. Thirteen isolates proved to be a mixture of different genotypes. Among the 149 pure isolates, 124 different genotypes could be recognized. The discriminatory power (D) for the individual markers ranged from 0.657 to 0.954. The D value of the panel of nine markers combined was 0.997. The multiplex multicolor approach was instrumental in rapid typing of a large number of isolates. There was no correlation between genotype and the clinical presentation of the infection. CONCLUSIONS/SIGNIFICANCE: There is a large genotypic diversity in clinical A. flavus isolates from India. The presence of more than one genotype in clinical samples illustrates the possibility that persons may be colonized by multiple genotypes and that any isolate from a clinical specimen is not necessarily the one actually causing infection. Microsatellites are excellent typing targets for discriminating between A. flavus isolates from various origins

PathogenMip Assay: A Multiplex Pathogen Detection Assay

The Molecular Inversion Probe (MIP) assay has been previously applied to a large-scale human SNP detection. Here we describe the PathogenMip Assay, a complete protocol for probe production and applied approaches to pathogen detection. We have demonstrated the utility of this assay with an initial set of 24 probes targeting the most clinically relevant HPV genotypes associated with cervical cancer progression. Probe construction was based on a novel, cost-effective, ligase-based protocol. The assay was validated by performing pyrosequencing and Microarray chip detection in parallel experiments. HPV plasmids were used to validate sensitivity and selectivity of the assay. In addition, 20 genomic DNA extracts from primary tumors were genotyped with the PathogenMip Assay results and were in 100% agreement with conventional sequencing using an L1-based HPV genotyping protocol. The PathogenMip Assay is a widely accessible protocol for producing and using highly discriminating probes, with experimentally validated results in pathogen genotyping, which could potentially be applied to the detection and characterization of any microbe

Single-Dose Intravenous Toxicity Study of IRDye 800CW in Sprague-Dawley Rats

Objective: Fluorophore-labeled contrast imaging agents are moving toward clinical use for a number of applications. The near-infrared dye IRDye 800CW is frequently used in its N-hydroxysuccinamide (NHS) ester form for labeling these agents. Following conjugation or breakdown of a labeled ligand, excess NHS ester is converted to the carboxylate form. To prepare for clinical use as a near-infrared fluorophore, a toxicity study was conducted on IRDye 800CW carboxylate. Methods: Male and female Sprague–Dawley rats were given a single intravenous or intradermal administration of IRDye 800CW carboxylate; Indocyanine Green was used as a comparative control. Animals were injected with varying doses of the test and control articles and observed for up to 14 days. Clinical chemistry, hematological, and pharmacokinetic analyses were performed on subgroups of animals. Organs were analyzed for content of the test article. Tissues were analyzed microscopically for pathological changes. Results: Based on hematologic, clinical chemistry, and histopathologic evaluation, single administration of IRDye 800CW carboxylate intravenously at dose levels of 1, 5, and 20 mg/kg or 20 mg/kg intradermally produced no pathological evidence of toxicity. Conclusion: A dose of 20 mg/kg was identified as the no observed adverse effect level following IV or ID routes of administration of IRDye 800CW

Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays

The diagnostic value of CRP, IL-8, PCT, and sTREM-1 in the detection of bacterial infections in pediatric oncology patients with febrile neutropenia

In this study, we evaluated C-reactive protein (CRP), interleukin (IL)-8, procalcitonin (PCT), and soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as predictors for bacterial infection in febrile neutropenia, plus their usefulness in febrile neutropenia during chemotherapy-induced gastrointestinal mucositis. Plasma was obtained from pediatric oncology patients at presentation with febrile neutropenia (n = 43) and 24-48 h later (n = 17). The patients were classified as having or not having a bacterial infection. Plasma was also obtained of patients in the absence and in the presence of mucositis (n = 26). At presentation with febrile neutropenia, median IL-8 and PCT levels were significantly increased in patients with a bacterial infection, in contrast to CRP and sTREM-1. IL-8 was the most sensitive marker for the early detection of bacterial infection, in combination with clinical parameters or PCT the sensitivity reached 100%. After 24-48 h, only PCT was significantly elevated during bacterial infection. IL-8 levels were significantly increased during mucositis. Mucositis did not cause considerable changes in PCT levels. IL-8 is the most useful marker for the early detection of bacterial infections, compared with CRP, PCT, and sTREM-1. IL-8 in combination with clinical parameters or PCT might be even more useful. Gastrointestinal mucositis alone does not affect PCT levels, in contrast to IL-8 levels, and therefore, PCT might be more useful for the detection of bacterial infections during mucositis than IL-8

NIOX VERO: Individualized Asthma Management in Clinical Practice

As we move toward an era of precision medicine, novel biomarkers of disease will enable the identification and personalized treatment of new endotypes. In asthma, fractional exhaled nitric oxide (FeNO) serves as a surrogate marker of airway inflammation that often correlates with the presence of sputum eosinophils. The increase in FeNO is driven by an upregulation of inducible nitric oxide synthase (iNOS) by cytokines, which are released as a result of type-2 airway inflammation. Scientific evidence supports using FeNO in routine clinical practice. In steroid-naive patients and in patients with mild asthma, FeNO levels decrease within days after corticosteroid treatment in a dose-dependent fashion and increase after steroid withdrawal. In difficult asthma, FeNO testing correlates with anti-inflammatory therapy compliance. Assessing adherence by FeNO testing can remove the confrontational aspect of questioning a patient about compliance and change the conversation to one of goal setting and ways to improve disease management. However, the most important aspect of incorporating FeNO in asthma management is the reduction in the risk of exacerbations. In a recent primary care study, reduction of exacerbation rates and improved symptom control without increasing overall inhaled corticosteroid (ICS) use were demonstrated when a FeNO-guided anti-inflammatory treatment algorithm was assessed and compared to the standard care. A truly personalized asthma management approach—showing reduction of exacerbation rates, overall use of ICS and neonatal hospitalizations—was demonstrated when FeNO testing was applied as part of the treatment algorithm that managed asthma during pregnancy. The aim of this article is to describe how FeNO and the NIOX VERO® analyzer can help to optimize diagnosis and treatment choices and to aid in the monitoring and improvement of clinical asthma outcomes in children and adults

Serological Markers for Inflammatory Bowel Disease in AIDS Patients with Evidence of Microbial Translocation

Background: Breakdown of the gut mucosal barrier during chronic HIV infection allows translocation of bacterial products such as lipopolysaccharides (LPS) from the gut into the circulation. Microbial translocation also occurs in inflammatory bowel disease (IBD). IBD serological markers are useful in the diagnosis of IBD and to differentiate between Crohn's disease (CD) and ulcerative colitis (UC). Here, we evaluate detection of IBD serological markers in HIV-infected patients with advanced disease and their relationship to HIV disease markers.Methods IBD serological markers (ASCA, pANCA, anti-OmpC, and anti-CBir1) were measured by ELISA in plasma from AIDS patients (n = 26) with low CD4 counts (<300 cells/$\mu$l) and high plasma LPS levels, and results correlated with clinical data. For meta-analysis, relevant data were abstracted from 20 articles. Results: IBD serological markers were detected in approximately 65% of AIDS patients with evidence of microbial translocation. An antibody pattern consistent with IBD was detected in 46%; of these, 75% had a CD-like pattern. Meta-analysis of data from 20 published studies on IBD serological markers in CD, UC, and non-IBD control subjects indicated that IBD serological markers are detected more frequently in AIDS patients than in non-IBD disease controls and healthy controls, but less frequently than in CD patients. There was no association between IBD serological markers and HIV disease markers (plasma viral load and CD4 counts) in the study cohort. Conclusions: IBD serological markers may provide a non-invasive approach to monitor HIV-related inflammatory gut disease. Further studies to investigate their clinical significance in HIV-infected individuals are warranted