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Abstract Background The prevalence and death rate arising from malaria infection, and emergence of other diseases showing similar symptoms to malaria require the development of malaria-specific and sensitive devices for its diagnosis. To address this, the design and fabrication of low-cost, rapid, paper-based analytical devices (µPAD) using surface-immobilized aptamers to detect the presence of a recombinant malarial biomarker—Plasmodium falciparum lactate dehydrogenase (rPfLDH)—is reported in this study. Methods Test zones on paper surfaces were created by covalently immobilizing streptavidin to the paper, subsequently attaching biotinylated aptamers to streptavidin. Aptamers selectively bound rPfLDH. The measurement of captured rPfLDH enzyme activity served as the means of detecting this biomarker. Enzyme activity across three replicate sensors was digitally quantified using the colorimetric Malstat assay. Results Screening of several different aptamers reported in the literature showed that aptamers rLDH7 and 2008s immobilized in this manner specifically recognised and captured PfLDH. Using rLDH7, the sensitivity of the µPAD sensor was evaluated and the µPAD sensor was applied for preferential detection of rPfLDH, both in buffered solutions of the protein and in spiked serum and red blood cell lysate samples. In buffered solutions, the test zone of the µPAD sensor exhibited a K D of 24 ± 11 nM and an empirical limit of detection of 17 nM, respectively, a limit similar to commercial antibody-based sensors exposed to rPfLDH. The specific recognition of 133 nM rPfLDH in undiluted serum and blood samples was demonstrated by the µPAD. Conclusion The reported µPAD demonstrates the potential of integrating aptamers into paper-based malarial rapid diagnostic tests. Graphical Abstract The assembly of µPAD sensors using APTEC assay principles for the detection the malarial biomarker, lactate dehydrogenase enzymes from Plasmodium falciparum (PfLDH). The aptamers immobilized at the test zones capture the PfLDH in samples. After washing the unbound sample components from the zones, Malstat assay reagents are added for colour development, proportional to the amount of captured PfLDH.

Background: The prevalence and death rate arising from malaria infection, and emergence of other diseases showing similar symptoms to malaria require the development of malaria-specific and sensitive devices for its diagnosis. To address this, the design and fabrication of low-cost, rapid, paper-based analytical devices (µPAD) using surface-immobilized aptamers to detect the presence of a recombinant malarial biomarker—Plasmodium falciparum lactate dehydrogenase (rPfLDH)—is reported in this study. Methods: Test zones on paper surfaces were created by covalently immobilizing streptavidin to the paper, subsequently attaching biotinylated aptamers to streptavidin. Aptamers selectively bound rPfLDH. The measurement of captured rPfLDH enzyme activity served as the means of detecting this biomarker. Enzyme activity across three replicate sensors was digitally quantified using the colorimetric Malstat assay. Results: Screening of several different aptamers reported in the literature showed that aptamers rLDH7 and 2008s immobilized in this manner specifically recognised and captured PfLDH. Using rLDH7, the sensitivity of the µPAD sensor was evaluated and the µPAD sensor was applied for preferential detection of rPfLDH, both in buffered solutions of the protein and in spiked serum and red blood cell lysate samples. In buffered solutions, the test zone of the µPAD sensor exhibited a KD of 24 ± 11 nM and an empirical limit of detection of 17 nM, respectively, a limit similar to commercial antibody-based sensors exposed to rPfLDH. The specific recognition of 133 nM rPfLDH in undiluted serum and blood samples was demonstrated by the µPAD. Conclusion: The reported µPAD demonstrates the potential of integrating aptamers into paper-based malarial rapid diagnostic tests. [ABSTRACT FROM AUTHOR]

Background: The objective of this study to detect Plasmodium and a subspecies of Plasmodium using filter paper in malaria endemic province, Sanliurfa, in Turkey, compare the results of nested PCR (nPCR) with microscopy for the diagnosis of malaria and present the epidemiological data of malaria., Methods: This study was carried out in malaria-endemic Sanliurfa between 2008 and 2011. Finger prick blood samples, thick and thin Giemsa-stained blood smears, were collected from 153 malaria-suspected farmworkers. The Giemsa-stained blood smears were examined microscopically. The obtained DNA products, extracted from blood-spotted filter papers or from the thick blood smears, were analysed by nPCR to amplify the 18S ssrRNA Plasmodium gene with genus and specific primers. The results of the microscopy were compared to the nPCR results., Results: Of the specimens, 7.2 % were determined as Plasmodium-positive by microscopy, whereas 9.8 % were determined as Plasmodium-positive by nPCR. Of the positive Plasmodium specimens, 93.33 % were identified as P. vivax. Four out of the 15 specimens that were microscopically diagnosed as negative were Plasmodium-positive with nPCR. When compared to the microscopy, the sensitivity, specificity, and positive predictive values of the nPCR were determined as 100, 97.2 and 73.3 %, respectively. nPCR was determined to be more sensitive and specific than microscopy., Conclusions: This study revealed that the accurate diagnosis of malaria by nPCR was compulsory in malaria-endemic Sanliurfa and nPCR should be applied routinely in laboratory studies.

Background: Isothermal amplification techniques are emerging as a promising method for malaria diagnosis since they are capable of detecting extremely low concentrations of parasite target while mitigating the need for infrastructure and training required by other nucleic acid based tests. Recombinase polymerase amplification (RPA) is promising for further development since it operates in a short time frame (<30 min) and produces a product that can be visually detected on a lateral flow dipstick. A self-sealing paper and plastic system that performs both the amplification and detection of a malaria DNA sequence is presented., Methods: Primers were designed using the NCBI nBLAST tools and screened using gel electrophoresis. Paper and plastic devices were prototyped using commercial design software and parts were cut using a laser cutter and assembled by hand. Synthetic copies of the Plasmodium 18S gene were spiked into solution and used as targets for the RPA reaction. To test the performance of the device the same samples spiked with synthetic target were run in parallel both in the paper and plastic devices and using conventional bench top methods., Results: Novel RPA primers were developed that bind to sequences present in the four species of Plasmodium which infect humans. The paper and plastic devices were found to be capable of detecting as few as 5 copies/µL of synthetic Plasmodium DNA (50 copies total), comparable to the same reaction run on the bench top. The devices produce visual results in an hour, cost approximately $1, and are self-contained once the device is sealed., Conclusions: The device was capable of carrying out the RPA reaction and detecting meaningful amounts of synthetic Plasmodium DNA in a self-sealing and self-contained device. This device may be a step towards making nucleic acid tests more accessible for malaria detection.