Eugen Ermantraut

HIV Load Testing with Small Samples of Whole Blood

ABSTRACT Access to human immunodeficiency virus (HIV) viral load (VL) testing is of paramount importance for the success of antiretroviral therapy treatment campaigns throughout the world. In many countries, limited laboratory infrastructure and transport capacities preclude a substantial number of people infected with HIV from accessing the necessary testing. Point-of-care diagnostic testing methods for those with HIV infection provide a compelling solution to addressing this challenge. To facilitate ease of use in such tests, finger-stick whole blood (WB) would constitute an ideal sample type if test performance equivalent to laboratory testing could be ensured. To determine the diagnostic sensitivity of a VL assay based on small volumes of WB, we analyzed 1,094 sample pairs of 1 ml of plasma and 10 μl of WB from donors confirmed to be HIV positive. The probability of detecting HIV nucleic acids in 10 μl of blood was 59.3% (95% confidence interval, 54.9 to 63.6%), 85.1% (80.0 to 90.2%), 91.5% (88.1 to 95%), and 100% when the corresponding plasma samples had an undetectable VL, a detectable VL less than 40 viral copies/ml (cp/ml), a VL between 40 and 4,000 cp/ml, and a VL greater than 4,000 cp/ml, respectively. Capillary blood and venous blood yielded comparable diagnostic sensitivities. Furthermore, our data indicate that WB could be used to monitor VL changes after highly active antiretroviral therapy (HAART) started. Thus, we have demonstrated the feasibility of small volumes of venous and finger-stick WB as valid samples for VL testing. This approach should facilitate the development of robust point-of-care HIV VL tests.

Competitive reporter monitored amplification (CMA)--quantification of molecular targets by real time monitoring of competitive reporter hybridization.

Background State of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests. Methodology and Principal Findings The described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique. Conclusions and Significance The system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests.

Building Highly Diverse Arrayed Substance Libraries by Micro Offset Printing

A unique method for the parallel miniaturised generation of microarrays of synthesized and/or immobilised material on wafersized substrates employing standard chemical protocols has been developed. The method is employed for the production of oligonucleotide-arrays and nanoELISA-assays.

Hepatitis C Virus RNA Quantitation in Venous and Capillary Small-Volume Whole-Blood Samples

ABSTRACT Quantitation of hepatitis C virus (HCV) RNA in plasma and serum samples is a costly procedure in both time and reagents. Additionally, cell-associated viral RNA may not be detected. This study evaluated the accuracy of HCV RNA quantitation in small-volume whole-blood (WB) samples, which would be appropriate for point-of-care diagnostic devices. HCV RNA was extracted from 222 clinical plasma and WB samples of 82 patients with chronic hepatitis C by a specific locked nucleic acid-mediated capture method and quantified by real-time reverse transcription-PCR. The results were compared to the reference plasma viral load determined with the COBAS AmpliPrep/TaqMan (CAP/CTM) HCV test. This assay had an analytical sensitivity of 9 IU per 10-μl sample (95% limit of detection [95% LOD]), a linearity range of 500 to 5 × 106 IU/ml, and was accurate in testing 10 HCV subtypes (<0.22 log10 unit) in plasma. The assay was matrix equivalent for plasma and WB samples (coefficient of determination [R2] of 0.943) and had a specificity of 100% (n = 20) in WB samples. The HCV RNA concentration in clinical WB samples exceeded the estimated hematocrit-corrected plasma viral loads by 0.22 log10 unit, but absolute quantitation results in plasma and WB samples were identical (95% confidence interval, −0.06 to 0.04 log10 unit). The sensitivity in WB samples was 100% (n = 141) for plasma concentrations above the 95% LOD. Quantitation results in 10-μl WB samples correlated linearly with the CAP/CTM HCV plasma test results (R2 = 0.919; n = 140) and did not differ between capillary and venous samples (R2 = 0.960; n = 40). This study shows that HCV RNA quantitation in 10-μl WB samples is appropriate for monitoring viral loads of >900 IU/ml, although the use of WB does not increase the diagnostic sensitivity.

Miniaturized Electrocaloric Flow Controller for Analyte Multiplexing and Cell/Particle Sorting

In the growing field of microsystems for analysis systems there is a major need of simple and stable micro devices to manipulate and switch fluid currents. Common devices use movable parts like membranes to control the flow through a given micropart. The main disadvantages of these microvalves are their liability to dirt, their cross sensitivity against external pressure differences and the difficulties to introduce the power to move the membranes. To circumvent the use of movable parts, different technical principles like the electroosmotic flow were introduced in the microworld. Microdevices using these technical systems often suffer from principle limitations too.

Nano Titer Plates: Micro Compartment Arrays for Biotechnological Applications

Arrays of micro compartments for combinatorial chemistry and biotechnological applications have been fabricated by means of photolithography and anisotropic chemical wet etching. Integration levels of 16′000 per 4″ wafer have been achieved. Optically transparent membranes or micro sieves allowing for rinsing between process steps can be incorporated. For the investigation of thermally activated biochemical reactions a micro compartment array with integrated thermo control has been developed, which allows the adjustment of thermal gradients over a 4″ wafer. Peak temperatures exceeding 90 °C at the center of the wafer as well as temperature gradients with ΔT = 40 °C could be demonstrated. To avoid fluidic crosstalking between adjacent compartments a technique has been developed, that allows selectively hydrophobizing the rims of the compartments.

Generation of libraries by print technologies

An efficient production of nucleic acid arrays on chips and their applications will open a great potential for biological key indications, such as early diagnostics, diagnostics and therapy monitoring of infections diseases or cancer, diagnostics of genetic diseases, predisposition diagnostics, monitoring of therapeutical principles (gene therapy), etc. However, for a production of thousands of such arrays, appropriate means of oligonucleotide synthesis on various solid supports will be required. Unfortunately, only a few successful ways have been described so far.

Integrated Device for Point-of-Care Genotyping

A new integrated microdevice is presented which allows the performance of complete arraybased genotyping assays within a single reactor.