Spencer W. Redding

Nano-bio-chips for high performance multiplexed protein detection: Determinations of cancer biomarkers in serum and saliva using quantum dot bioconjugate labels

The integration of semiconductor nanoparticle quantum dots (QDs) into a modular, microfluidic biosensor for the multiplexed quantitation of three important cancer markers, carcinoembryonic antigen (CEA), cancer antigen 125 (CA125), and Her-2/Neu (C-erbB-2) was achieved. The functionality of the integrated sample processing, analyte capture and detection modalities was demonstrated using both serum and whole saliva specimens. Here, nano-bio-chips that employed a fluorescence transduction signal with QD-labeled detecting antibody were used in combination with antigen capture by a microporous agarose bead array supported within a microfluidics ensemble so as to complete the sandwich-type immunoassay. The utilization of QD probes in this miniaturized biosensor format resulted in signal amplification 30 times relative to that of standard molecular fluorophores as well as affording a reduction in observed limits of detection by nearly 2 orders of magnitude (0.02 ng/mL CEA; 0.11 pM CEA) relative to enzyme-linked immunosorbent assay (ELISA). Assay validation studies indicate that measurements by the nano-bio-chip system correlate to standard methods at R(2)=0.94 and R(2)=0.95 for saliva and serum, respectively. This integrated nano-bio-chip assay system, in tandem with next-generation fluorophores, promises to be a sensitive, multiplexed tool for important diagnostic and prognostic applications.

Use of saliva-based nano-biochip tests for acute myocardial infarction at the point of care: A feasibility study

BACKGROUND For adults with chest pain, the electrocardiogram (ECG) and measures of serum biomarkers are used to screen and diagnose myocardial necrosis. These measurements require time that can delay therapy and affect prognosis. Our objective was to investigate the feasibility and utility of saliva as an alternative diagnostic fluid for identifying biomarkers of acute myocardial infarction (AMI). METHODS We used Luminex and lab-on-a-chip methods to assay 21 proteins in serum and unstimulated whole saliva procured from 41 AMI patients within 48 h of chest pain onset and from 43 apparently healthy controls. Data were analyzed by use of logistic regression and area under curve (AUC) for ROC analysis to evaluate the diagnostic utility of each biomarker, or combinations of biomarkers, in screening for AMI. RESULTS Both established and novel cardiac biomarkers demonstrated significant differences in concentrations between patients with AMI and controls without AMI. The saliva-based biomarker panel of C-reactive protein, myoglobin, and myeloperoxidase exhibited significant diagnostic capability (AUC = 0.85, P < 0.0001) and in conjunction with ECG yielded strong screening capacity for AMI (AUC = 0.96) comparable to that of the panel (brain natriuretic peptide, troponin-I, creatine kinase-MB, myoglobin; AUC = 0.98) and far exceeded the screening capacity of ECG alone (AUC approximately 0.6). En route to translating these findings to clinical practice, we adapted these unstimulated whole saliva tests to a novel lab-on-a-chip platform for proof-of-principle screens for AMI. CONCLUSIONS Complementary to ECG, saliva-based tests within lab-on-a-chip systems may provide a convenient and rapid screening method for cardiac events in prehospital stages for AMI patients.

The use of glucocorticosteroids to lessen the inflammatory sequelae following third molar surgery

Acceptance of the use of glucocorticosteroids in density to control postsurgical inflammation has been impaired by concerns over side effects, adrenal suppression, and efficacy. The pattern of administration generally used is characterized as short-term, high-dose or pulse therapy, which has not been associated with significant side effects or adrenal suppression beyond 10 days. The selection of an appropriate glucocorticosteroid with minimal mineralocorticoid activity and extended biological activity is desirable. Oral and parenteral dosing are possible, and the latter can be administered as acetates (repository) or esters. The efficacy of glucocorticosteroids in reducing pain, swelling, and trismus after third molar surgery is difficult to ascertain because of methodological inconsistencies between investigations. In general, studies that used low dosing schedules have failed to produce dramatic and prolonged results. High-dosing intravenous (IV) studies have demonstrated significant short-term improvements, but the effects were not sustained. Combining IV administration with multiple oral dosing or a single intramuscular (IM) dose may be required to extend short-term improvement. High-dosing IM studies have shown significant and sustained anti-inflammatory effects with a single dose administered either pre- or post-operatively.

Inhibition of Candida albicans biofilm formation on denture material

OBJECTIVE The aim was to determine the ability of several thin-film polymer formulations, with and without incorporated antifungals, to inhibit Candida albicans biofilm growth on denture material. The inhibition of C. albicans biofilms on maxillary dentures could play a significant role in preventing the development of denture stomatitis. STUDY DESIGN Low-porosity and high-porosity thin-film polymer formulations were used and one of the following fungicides was added: 1) chlorhexidine diacetate at 1.0%; 2) nystatin at 1.0%; or 3) amphotericin B at 0.1%. These coatings were placed on rectangular (12 x 10 mm) dental resin material samples. A subset of the coated dental materials were brushed to simulate denture cleaning for 1 minute per day for 1 year. Candida albicans biofilms were formed on polymethylmethacrylate (PMMA) specimens placed in 24-well polystyrene plates, and the extent of biofilm formation on coated and noncoated specimens was assessed using a 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay. RESULTS Thin-film polymer PMMA coatings alone, without an antifungal agent, produced a small significant reduction in C. albicans biofilm formation compared with control PMMA. However, incorporation of antifungal medications into the thin-film polymer reduced biofilm formation between 70% and 80% with nystatin, and between 50% and 60% with amphotericin B. Biofilm reduction with chlorhexidine (up to 98%) was significantly greater than all other formulations tested (P < .025). CONCLUSION This novel thin-film coating with various antifungals effectively inhibits C. albicans biofilm formation and should be evaluated as a potential preventive therapy for denture stomatitis.

Multiple patterns of resistance to fluconazole in Candida glabrata isolates from a patient with oropharyngeal candidiasis receiving head and neck radiation

ABSTRACT Candida glabrata has emerged in recent years as a significant cause of systemic fungal infection. We have previously reported on the first three patients receiving radiation for head and neck cancer to develop oropharyngeal candidiasis due to C. glabrata. The goal of this study was to track the development of increased fluconazole resistance in C. glabrata isolates and to evaluate previously described genetic mechanisms associated with this resistance from one of these three patients. The patient was a 52-year-old man with squamous cell carcinoma treated with radiation. At week 7 of his radiation, he developed oropharyngeal candidiasis, which was treated with 200 mg of fluconazole daily for 2 weeks. Serial cultures from this and three subsequent time points yielded C. glabrata. Isolates from these cultures were subjected to antifungal susceptibility testing, DNA karyotyping, and evaluation of the expression of genes previously associated with C. glabrata resistance to fluconazole, CgCDR1, CgCDR2, and CgERG11. Two strains (A and B) of C. glabrata were identified and found to display different patterns of resistance development and gene expression. Strain A developed resistance over a 2-week period and showed no overexpression of these genes. In contrast, strain B first showed resistance 6 weeks after fluconazole therapy was discontinued but showed overexpression of all three genes. In conclusion, development of resistance to fluconazole by C. glabrata is a highly varied process involving multiple molecular mechanisms.

Clinical Evaluation and Microbiology of Oropharyngeal Infection Due to Fluconazole-Resistant Candida in Human Immunodeficiency Virus-Infected Patients

Signs and symptoms of oropharyngeal candidiasis (OPC) were correlated with microbiology and clinical response to fluconazole in a cohort of patients with advanced human immunodeficiency virus (HIV) infection and recurrent OPC. Sixty-four HIV-infected patients with a median CD4 cell count of < 50/mm3 (range, 3-318/mm3) who presented with OPC were enrolled in a longitudinal study. Specimens for cultures were taken weekly until clinical resolution. Therapy with fluconazole was increased weekly as required to a maximum daily dose of 800 mg until resolution of symptoms and oral lesions. Resistant or dose-dependent susceptible yeasts, defined as a minimum inhibitory concentration of > or = 16 micrograms/mL, were detected in 48 (31%) of 155 episodes. Clinical resolution with fluconazole therapy occurred in 107 (100%) of 107 episodes with susceptible yeasts vs. 44 (92%) of 48 episodes with resistant or dose-dependent susceptible strains (P = .008). Patients from whom fluconazole-resistant yeasts were isolated required longer courses of therapy and higher doses of fluconazole for response, but overall, excellent responses to fluconazole were seen in patients with advanced HIV infection.

A randomized trial of continuous or intermittent therapy with fluconazole for oropharyngeal candidiasis in HIV-infected patients: clinical outcomes and development of fluconazole resistance

PURPOSE The effects of continuous or intermittent therapy with fluconazole on the recurrence of and the development of fluconazole resistance are not known. PATIENTS AND METHODS We studied human immunodeficiency virus (HIV)-positive patients with CD4 cell count <350 x 10(6)/L and oropharyngeal candidiasis in a prospective, randomized study. After initial treatment, 20 patients (16 of whom completed 3 months of follow-up) received continuous fluconazole at 200 mg/day, and 48 patients (28 of whom completed follow-up) received intermittent therapy at the time of symptomatic relapses. Oral samples were obtained weekly during episodes of infection and quarterly as surveillance cultures. Development of resistance was defined as a fourfold rise in minimum inhibitory concentration (MIC) to at least 16 microg/mL from the initial culture in the same species, the emergence of new, resistant (MIC > or =16 microg/mL) species, or a significant increase in the proportion of resistant isolates. RESULTS During a mean follow-up of 11 months, median annual relapse rates were lower in patients on continuous therapy (0 episodes/year) than in patients on intermittent therapy (4.1 episodes/year; P <0.001). Sterile cultures were seen in 6 of 16 (38%) patients on continuous therapy compared with 3 of 28 (11%) on intermittent therapy (P = 0.04). Microbiological resistance developed in 9 of 16 (56%) patients on continuous treatment, compared with 13 of 28 (46%) on intermittent treatment (P = 0.75). However, despite isolates with increased MICs, 42 of 44 patients responded to fluconazole in doses up to 800 mg/day. CONCLUSIONS In patients with frequent recurrences, continuous suppressive therapy significantly reduced relapses and colonization. Resistance occurred with both continuous and intermittent therapy; however, therapeutic responses were excellent.

Nano-bio-chip sensor platform for examination of oral exfoliative cytology

Oral cancer is a deadly and disfiguring disease that could greatly benefit from new diagnostic approaches enabling early detection. In this pilot study, we describe a nano-bio-chip (NBC) sensor technique for analysis of oral cancer biomarkers in exfoliative cytology specimens, targeting both biochemical and morphologic changes associated with early oral tumorigenesis. Here, oral lesions from 41 dental patients, along with normal epithelium from 11 healthy volunteers, were sampled using a noninvasive brush biopsy technique. Specimens were enriched, immunolabeled, and imaged in the NBC sensor according to previously established assays for the epidermal growth factor receptor (EGFR) biomarker and cytomorphometry. A total of 51 measurement parameters were extracted using custom image analysis macros, including EGFR labeling intensity, cell and nuclear size, and the nuclear-to-cytoplasmic ratio. Four key parameters were significantly elevated in both dysplastic and malignant lesions relative to healthy oral epithelium, including the nuclear area and diameter (P < 0.0001), the nuclear-to-cytoplasmic ratio (P < 0.0001), and EGFR biomarker expression (P < 0.03). Further examination using logistic regression and receiver operating characteristic curve analyses identified morphologic features as the best predictors of disease (area under the curve ≤0.93) individually, whereas a combination of all features further enhanced discrimination of oral cancer and precancerous conditions (area under the curve, 0.94) with high sensitivity and specificity. Further clinical trials are necessary to validate the regression model and evaluate other potential biomarkers, but this pilot study supports the NBC sensor technique as a promising new diagnostic tool for early detection of oral cancer, which could enhance patient care and survival. Cancer Prev Res; 3(4); 518–28. ©2010 AACR.

Candida glabrata Oropharyngeal Candidiasis in Patients Receiving Radiation Treatment for Head and Neck Cancer

ABSTRACT Candida glabrata colonization is common in patients receiving radiation treatment for head and neck cancer, but to our knowledge has never been described as the infecting organism with oropharyngeal candidiasis (OPC). This study presents the first three patients described with C. glabrata OPC in this patient population. Patient 1 developed C. glabrata OPC and required fluconazole, 800 mg/day, for clinical resolution. Antifungal susceptibility testing revealed a MIC of fluconazole of >64 μg/ml. Elapsed time from initial culturing to treatment decision was 7 days. Patients 2 and 3 developed C. glabrata OPC. They were patients in a study evaluating OPC infections, and cultures were taken immediately. CHROMagar Candida plates with 0, 8, and 16 μg of fluconazole/ml were employed for these cultures. Lavender colonies, consistent with C. glabrata, grew on the 0- and 8-μg plates but not on the 16-μg plate from patient 2 and grew on all three plates from patient 3. Based on these data, a fluconazole dose of 200 mg/day was chosen for patient 2 and a dose of 400 mg/day was chosen for patient 3, with clinical resolution in both. Elapsed time from initial culturing to treatment decision was 2 days. C. glabrata does cause OPC in head and neck radiation treatment patients, and the use of fluconazole-impregnated chromogenic agar may significantly reduce treatment decision time compared to that with conventional culturing and antifungal susceptibility testing.

Oropharyngeal candidiasis caused by non-albicans yeast in patients receiving external beam radiotherapy for head-and-neck cancer

PURPOSE To characterize non-albicans Candida oral infections in patients with head-and-neck cancer receiving external beam radiotherapy (EBRT) with or without concurrent chemotherapy. METHODS AND MATERIALS Thirty-seven patients with head-and-neck cancer received EBRT in 2.0-Gy daily fractions to a median dose of 60.4 Gy (range 38-82.8, mean 64.6). They were followed for oropharyngeal candidiasis (OPC) confirmed by positive examination, positive KOH smear, and/or positive swab or swish culture. Samples were identified and plated on chromogenic media to identify non-albicans yeasts. Colonies were plated on Sabouraud dextrose slants for microdilution antifungal susceptibility testing to fluconazole. DNA typing, including karyotyping, restriction fragment length polymorphism analysis, and Southern blot hybridization with the moderately repetitive Ca3 probe, was performed on selected isolates to confirm individual species. RESULTS Of the 37 patients, 10 (27%) developed OPC, and 26 (70.3%) displayed Candida carriage state. The median EBRT dose at time of positive culture was 22.5 Gy and at time of OPC was 28.6 Gy. Of the 6 patients receiving chemotherapy and EBRT, 4 (66%) developed OPC at median dose of 27.6 Gy. Three (8%) of 37 patients were infected with non-albicans Candida, and 3 (30%) of all 10 infections were caused by these organisms. CONCLUSION Non-albicans Candida is emerging as a relatively common cause of OPC in head-and-neck cancer patients. Chromogenic media are helpful to screen these infections. Our data also suggest a greater likelihood of developing OPC in patients receiving concomitant chemotherapy and EBRT.