BackgroundMore than 35 million people in developing countries are living with HIV infection. An enormous global effort is now underway to bring antiretroviral treatment to at least 3 million of those infected. While drug prices have dropped considerably, the cost and technical complexity of laboratory tests essential for the management of HIV disease, such as CD4 cell counts, remain prohibitive. New, simple, and affordable methods for measuring CD4 cells that can be implemented in resource-scarce settings are urgently needed.Methods and FindingsHere we describe the development of a prototype for a simple, rapid, and affordable method for counting CD4 lymphocytes. Microliter volumes of blood without further sample preparation are stained with fluorescent antibodies, captured on a membrane within a miniaturized flow cell and imaged through microscope optics with the type of charge-coupled device developed for digital camera technology. An associated computer algorithm converts the raw digital image into absolute CD4 counts and CD4 percentages in real time. The accuracy of this prototype system was validated through testing in the United States and Botswana, and showed close agreement with standard flow cytometry (r = 0.95) over a range of absolute CD4 counts, and the ability to discriminate clinically relevant CD4 count thresholds with high sensitivity and specificity.ConclusionAdvances in the adaptation of new technologies to biomedical detection systems, such as the one described here, promise to make complex diagnostics for HIV and other infectious diseases a practical global reality.
In the last decade, saliva has been advocated as a non-invasive alternative to blood as a diagnostic fluid. However, use of saliva has been hindered by the inadequate sensitivity of current methods to detect the lower salivary concentrations of many constituents compared to serum. Furthermore, developments in the areas related to lab-on-a-chip systems for saliva-based point of care diagnostics are complicated by the high viscosity and heterogeneous properties associated with this diagnostic fluid. The biomarker C-reactive protein (CRP) is an acute phase reactant and a well-accepted indicator of inflammation. Numerous clinical studies have established elevated serum CRP as a strong, independent risk factor for the development of cardiovascular disease (CVD). CVD has also been associated with oral infections (i.e. periodontal diseases) and there is evidence that systemic CRP may be a link between the two. Clinical measurements of CRP in serum are currently performed with "high sensitivity" CRP (hsCRP) enzyme-linked immunosorbent assay (ELISA) tests that lack the sensitivity for the detection of this important biomarker in saliva. Because measurement of salivary CRP may represent a novel approach for diagnosing and monitoring chronic inflammatory disease, including CVD and periodontal diseases, the objective of this study was to apply an ultra-sensitive microchip assay system for the measurement of CRP in human saliva. Here, we describe this novel lab-on-a-chip system in its first application for the measurement of CRP in saliva and demonstrate its advantages over the traditional ELISA method. The increased sensitivity of the microchip system (10 pg ml(-1) of CRP with 1000-fold dilution of saliva sample) is attributed to its inherent increased signal to noise ratio, resulting from the higher bead surface area available for antigen/antibody interactions and the high stringency washes associated with this approach. Finally, the microchip assay system was utilized in this study to provide direct experimental evidence that chronic periodontal disease may be associated with higher levels of salivary CRP.
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).
Salivary secretions contain a variety of molecules that reflect important pathophysiological activities. Quantitative changes of specific salivary biomarkers could have significance in the diagnosis and management of both oral and systemic diseases. Modern point-of-care technologies with enhanced detection capabilities are needed to implement a significant advancement in salivary diagnostics. One such promising technology is the recently described lab-on-a-chip (LOC) assay system, in which assays are performed on chemically sensitized beads populated into etched silicon wafers with embedded fluid handling and optical detection capabilities. Using this LOC system, complex assays can be performed with small sample volumes, short analysis times, and markedly reduced reagent costs. This report describes the use of LOC methodologies to assess the levels of interleukin-1beta (IL-1beta), C-reactive protein (CRP), and matrix metalloproteinase-8 (MMP-8) in whole saliva, and the potential use of these biomarkers for diagnosing and categorizing the severity and extent of periodontitis. This study demonstrates that the results achieved by the LOC approach are in agreement with those acquired with standard enzyme-linked immunosorbent assay (ELISA), with significant IL-1beta and MMP-8 elevations in whole saliva of periodontitis patients. Furthermore, because of the superior detection capacities associated with the LOC approach, unlike those with ELISA, significant differences in CRP levels between periodontitis patients and normal subjects are observed. Finally, principal component analysis (PCA) is performed to yield an efficient method to discriminate between periodontally healthy and unhealthy patients, thus increasing the diagnostic value of these biomarkers for periodontitis when examined with the integrated LOC sensor system.
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