This work addresses the design of an Ebola diagnostic test involving a simple, rapid, specific and highly sensitive procedure based on isothermal amplification on magnetic particles with ...electrochemical readout. Ebola padlock probes were designed to detect a specific L-gene sequence present in the five most common Ebola species. Ebola cDNA was amplified by rolling circle amplification (RCA) on magnetic particles. Further re-amplification was performed by circle-to-circle amplification (C2CA) and the products were detected in a double-tagging approach using a biotinylated capture probe for immobilization on magnetic particles and a readout probe for electrochemical detection by square-wave voltammetry on commercial screen-printed electrodes. The electrochemical genosensor was able to detect as low as 200 ymol, corresponding to 120 cDNA molecules of L-gene Ebola virus with a limit of detection of 33 cDNA molecules. The isothermal double-amplification procedure by C2CA combined with the electrochemical readout and the magnetic actuation enables the high sensitivity, resulting in a rapid, inexpensive, robust and user-friendly sensing strategy that offers a promising approach for the primary care in low resource settings, especially in less developed countries.
•A novel genosensor for Ebola virus based on isothermal amplification on magnetic particles is presented.•Padlock probes were designed for the isothermal amplification of synthetic cDNA.•Electrochemical genosensing combined with circle-to-circle amplification provides an outstanding LOD in 2.5h.•The Ebola genosensor meets the demands for ASSURED diagnosis recommended by WHO.
In the PORTEC-3 trial, women with high-risk endometrial cancer (HR-EC) were randomised to receive pelvic radiotherapy (RT) with or without concurrent and adjuvant chemotherapy (two cycles of ...cisplatin 50 mg/m2 in weeks 1 and 4 of RT, followed by four cycles of carboplatin AUC5 and paclitaxel 175 mg/m2). Pathology review was required before patient enrolment. The aim of this analysis was to evaluate the role of central pathology review before randomisation.
A total of 1295 cases underwent pathology review to confirm HR-EC in the Netherlands (n = 395) and the UK (n = 900), and for 1226/1295 (95%) matching review and original reports were available. In total, 329 of these patients were enrolled in the PORTEC-3 trial: 145 in the Netherlands and 184 in the UK, comprising 48% of the total PORTEC-3 cohort of 686 participants. Areas of discrepancies were evaluated, and inter-observer agreement between original and review opinion was evaluated by calculating the kappa value (κ).
In the 1226 pathology reviews, 6356 selected items were evaluable for both original and review pathology. In 43% of cases at least one pathology item changed after review. For 102 patients (8%), this discrepancy led to ineligibility for the PORTEC-3 trial, most frequently due to differences in the assessment of histological type (34%), endocervical stromal involvement (27%) and histological grade (19%). Lowest inter-observer agreement was found for histological type (κ = 0.72), lymph-vascular space invasion (κ = 0.72) and histological grade (κ = 0.70).
Central pathology review by expert gynaeco-pathologists changed histological type, grade or other items in 43% of women with HR-EC, leading to ineligibility for the PORTEC-3 trial in 8%. Upfront pathology review is essential to ensure enrolment of the target trial-population, and to avoid over- or undertreatment, especially when treatment modalities with substantial toxicity are involved.
This study is registered with ISRCTN (ISRCTN14387080, www.controlled-trials.com) and with ClinicalTrials.gov (NCT00411138).
The Acquired Immune Deficiency Syndrome (AIDS) affects the life of millions of people around the world. Although rapid and low cost screening tests are widely available for the diagnosis of HIV ...infection, the count of CD4+ T lymphocytes remains a drawback in the areas mostly affected by the HIV, being this control imperative for assessing the deterioration of the immunological system and the progression towards AIDS, when the counting of cells falls down 200cellsμL−1. This paper describes a high-throughput, simple and rapid method for CD4+ T lymphocytes quantification, directly in whole blood, based on a magneto ELISA. The CD4 cells are separated and preconcentrated from whole blood in magnetic particles, and labeled with an enzyme for the optical readout performed with a standard microplate reader. The magneto ELISA is able to reach the whole CD4 counting range of medical interest, being the limit of detection as low as 50 CD4+ cells per μL of whole blood, without any pretreatment. This method is a highly suitable alternative diagnostic tool for the expensive flow cytometry at the community and primary care level, providing a sensitive method but by using instrumentation widely available in low-resource settings laboratories and requiring low-maintenance, as is the case of a microplate reader operated by filters.
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•CD4 magneto immunoassay for whole blood with a LOD of 50CD4cellsμL−1.•The test spans the clinical range for its application in AIDS diagnosis and following up.•The CD4 cells are preconcentrated from whole blood without any sample treatment.•The magneto immunoassay offers an alternative to flow cytometry in low resource settings.•This high-throughput test allows the detection of several samples in decentralized small health centers.
The counting of CD4+ T lymphocytes is a clinical parameter used for AIDS diagnosis and follow-up. As this disease is particularly prevalent in developing countries, simple and affordable CD4 cell ...counting methods are urgently needed in resource-limited settings. This paper describes an electrochemical magneto-actuated biosensor for CD4 count in whole blood. The CD4+ T lymphocytes were isolated, preconcentrated and labeled from 100μL of whole blood by immunomagnetic separation with magnetic particles modified with antiCD3 antibodies. The captured cells were labeled with a biotinylated antiCD4 antibody, followed by the reaction with the electrochemical reporter streptavidin-peroxidase conjugate. The limit of detection for the CD4 counting magneto-actuated biosensor in whole blood was as low as 44cellsμL−1 while the logistic range was found to be from 89 to 912cellsμL−1, which spans the whole medical interest range for CD4 counts in AIDS patients. The electrochemical detection together with the immunomagnetic separation confers high sensitivity, resulting in a rapid, inexpensive, robust, user-friendly method for CD4 counting. This approach is a promising alternative for the costly standard flow cytometry and suitable as diagnostic tool at decentralized practitioner sites in low resource settings, especially in less developed countries.
•There is an urgent need for novel affordable alternatives to flow cytometry for CD4 count to monitor the AIDS disease and the treatment in low resource settings.•An electrochemical magneto-actuated biosensor for CD4+ T lymphocytes count in whole blood is presented, with a LOD of 44cellsμL−1 covering the clinical range of CD4+ lymphocytes in AIDS diagnosis and follow up.•The electrochemical magneto biosensors offer an exciting alternative in resource-scarce settings, as rapid, cost-effective devices that can be handled for unskilled personnel at the community care level.
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