Rapid Molecular Assays for Specific Detection and Quantitation of Loa loa Microfilaremia

Loa loa is a filarial nematode that infects over 10 million people in Africa. Most infections cause no symptoms, but individuals with large numbers of blood-stage microfilariae are at risk for fatal reactions to ivermectin, an antiparasitic agent used to treat and prevent infections with Onchocerca volvulus, a related filarial parasite that may occur alongside L. loa. To address the urgent need for a point-of-care L. loa diagnostic assay, we screened a Loa microfilaria gene expression library and identified 18 Loa-specific DNA targets. From two targets, we developed a novel, rapid quantitative PCR assay for estimating L. loa microfilaria burden. The assay is highly sensitive (detects a single microfilaria in 20 µL of blood) and correlates well with microfilaria counts obtained with conventional microscopic techniques. The assay is species-specific for L. loa compared with related filarial parasites (including O. volvulus) and can be used in its current form in resource-rich areas as a diagnostic tool for L. loa infection. Although modifications will be required to make point-of-care use feasible, our assay provides a proof of concept for a potentially valuable tool to identify individuals at risk for adverse reactions to ivermectin and to facilitate the implementation of filarial control programs

Sample introduction interface for on-chip nucleic acid-based analysis of Helicobacter pylori from stool samples.

Despite recent advances in microfluidic-based integrated diagnostic systems, the sample introduction interface, especially with regards to large volume samples, has often been neglected. We present a sample introduction interface that allows direct on-chip processing of crude stool samples for the detection of Helicobacter pylori (H. pylori). The principle of IFAST (immiscible filtration assisted by surface tension) was adapted to include a large volume sample chamber with a septum-based interface for stool sample introduction. Solid chaotropic salt and dry superparamagnetic particles (PMPs) could be stored on-chip and reconstituted upon sample addition, simplifying the process of release of DNA from H. pylori cells and its binding to the PMPs. Finally, the PMPs were pulled via a magnet through a washing chamber containing an immiscible oil solution and into an elution chamber where the DNA was released into aqueous media for subsequent analysis. The entire process required only 7 min while enabling a 40-fold reduction in working volume from crude biological samples. The combination of a real-world interface and rapid DNA extraction offers the potential for the methodology to be used in point-of-care (POC) devices

Immobilization of antibodies in micropatterns for cell detection by optical diffraction

Optical diffraction at biochemically microstructured surfaces has been investigated for the label-free in situ detection of cells. The new sensor concept is based on regular arrays of covalently coupled antibodies, which selectively bind cells from solution. Due to the adsorption process, changes are imposed on the intensity distribution of the diffracted light, which can serve to quantify the amount of adsorbed cells. For the formation of such microstructures, different classical film preparation techniques were transferred to a mesoscopic scale by the use of microcontact printing (μCP). Alternatively, receptors were functionalized with thiol groups prior to the immobilization process and directly printed onto the gold surface. Compared to imprinting of non-functionalized proteins on gold, a better replication of the micropatterns could be obtained. Additionally, a significantly lower amount of defects was observed than for the classical coupling techniques. Using such microstructures, first experiments on the detection of Escherichia coli bacteria were performed. Diffraction patterns have been observed for concentrations equal or higher than 106 cells/ml. In time dependent experiments, diffraction spots occurred after 30 – 90 min or 10 – 20 min, depending on whether non-specific cell adsorption or specific binding to anti-E. coli IgG was studied. A first quantitative analysis of the diffraction patterns shows that the total amount of diffracted light increases with increasing incubation time

Heat pain detection threshold is associated with the area of secondary hyperalgesia following brief thermal sensitization: a study of healthy male volunteers

Morten Sejer Hansen,1 Jørn Wetterslev,2 Christian Bressen Pipper,3 Mohammad Sohail Asghar,1 Jørgen Berg Dahl4 1Department of Anesthesiology, 4231, Centre of Head and Orthopedics, Rigshospitalet, 2Copenhagen Trial Unit, Centre for Clinical Intervention Research, Department 7812, 3Section of Biostatistics, Faculty of Health, Copenhagen University, Copenhagen, 4Department of Anesthesiology, Department Z, Bispebjerg Hospital, Copenhagen, Denmark Introduction: The area of secondary hyperalgesia following brief thermal sensitization (BTS) of the skin and heat pain detection thresholds (HPDT) may both have predictive abilities in regards to pain sensitivity and clinical pain states. The association between HPDT and secondary hyperalgesia, however, remains unsettled, and the dissimilarities in physiologic properties suggest that they may represent 2 distinctively different pain entities. The aim of this study was to investigate the association between HPDT and BTS-induced secondary hyperalgesia. Methods: A sample of 121 healthy male participants was included and tested on 2 separate study days with BTS (45°C, 3 minutes), HPDT, and pain during thermal stimulation (45°C, 1 minute). Areas of secondary hyperalgesia were quantified after monofilament pinprick stimulation. The pain catastrophizing scale (PCS) and hospital anxiety and depression scale (HADS) were also applied. Results: A significant association between HPDT and the size of the area of secondary hyperalgesia (p<0.0001) was found. The expected change in area of secondary hyperalgesia due to a 1-degree increase in HPDT was estimated to be −27.38 cm2, 95% confidence interval (CI) of −37.77 to −16.98 cm2, with an R2 of 0.19. Likewise, a significant association between HADS-depression subscore and area of secondary hyperalgesia (p=0.046) was found, with an estimated expected change in secondary hyperalgesia to a 1-point increase in HADS-depression subscore of 11 cm2, 95% CI (0.19–21.82), and with R2 of 0.03. We found no significant associations between secondary hyperalgesia area and PCS score or pain during thermal stimulation. Conclusion: HPDT and the area of secondary hyperalgesia after BTS are significantly associated; however, with an R2 of only 19%, HPDT only offers a modest explanation of the inter-participant variation in the size of the secondary hyperalgesia area elicited by BTS. Keywords: pain, central nervous system sensitization, hyperalgesia, pain threshold, healthy volunteers, catastrophization, secondary hyperalgesia, central sensitizatio

Solvation of oligo(ethylene glycol)-terminated self-assembled monolayers studied by vibrational sum frequency spectroscopy

Self-assembled monolayers of methyl(1-mercaptoundec-11-yl)tri(ethylene glycol) (CH3O(C2H4O)3C11H22SH, EG3-OMe) adsorbed on gold were investigated by IR−vis sum frequency generation in the range of the C−H stretching vibrations. Comparison of the monolayers in ambient atmosphere, in contact with water, and in contact with carbon tetrachloride revealed that the film structure is strongly disturbed by the interaction of the liquid with the monolayer. The ordered structure found in air undergoes an amorphization upon exposure to the solvents. The experiments demonstrate that in situ analysis of the film structure is mandatory