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      In Silico Identification of Novel Biomarkers and Development of New Rapid Diagnostic Tests for the Filarial Parasites Mansonella perstans and Mansonella ozzardi

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          Abstract

          Mansonelliasis is a widespread yet neglected tropical infection of humans in Africa and South America caused by the filarial nematodes, Mansonella perstans, M. ozzardi, M. rodhaini and M. streptocerca. Clinical symptoms are non-distinct and diagnosis mainly relies on the detection of microfilariae in skin or blood. Species-specific DNA repeat sequences have been used as highly sensitive biomarkers for filarial nematodes. We have developed a bioinformatic pipeline to mine Illumina reads obtained from sequencing M. perstans and M. ozzardi genomic DNA for new repeat biomarker candidates which were used to develop loop-mediated isothermal amplification (LAMP) diagnostic tests. The M. perstans assay based on the Mp419 repeat has a limit of detection of 0.1 pg, equivalent of 1/1000 th of a microfilaria, while the M. ozzardi assay based on the Mo2 repeat can detect as little as 0.01 pg. Both LAMP tests possess remarkable species-specificity as they did not amplify non-target DNAs from closely related filarial species, human or vectors. We show that both assays perform successfully on infected human samples. Additionally, we demonstrate the suitability of Mp419 to detect M. perstans infection in Culicoides midges. These new tools are field deployable and suitable for the surveillance of these understudied filarial infections.

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          Most cited references 55

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          Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects.

          Loop-mediated isothermal amplification (LAMP), a newly developed gene amplification method, combines rapidity, simplicity, and high specificity. Several tests have been developed based on this method, and simplicity is maintained throughout all steps, from extraction of nucleic acids to detection of amplification. In the LAMP reaction, samples are amplified at a fixed temperature through a repetition of two types of elongation reactions occurring at the loop regions: self-elongation of templates from the stem loop structure formed at the 3'-terminal and the binding and elongation of new primers to the loop region. The LAMP reaction has a wide range of possible applications, including point-of-care testing, genetic testing in resource-poor settings (such as in developing countries), and rapid testing of food products and environmental samples.
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            Satellite DNAs between selfishness and functionality: structure, genomics and evolution of tandem repeats in centromeric (hetero)chromatin.

            Satellite DNAs (tandemly repeated, non-coding DNA sequences) stretch over almost all native centromeres and surrounding pericentromeric heterochromatin. Once considered as inert by-products of genome dynamics in heterochromatic regions, recent studies showed that satellite DNA evolution is interplay of stochastic events and selective pressure. This points to a functional significance of satellite sequences, which in (peri)centromeres may play some fundamental functional roles. First, specific interactions with DNA-binding proteins are proposed to complement sequence-independent epigenetic processes. The second role is achieved through RNAi mechanism, in which transcripts of satellite sequences initialize heterochromatin formation. In addition, satellite DNAs in (peri)centromeric regions affect chromosomal dynamics and genome plasticity. Paradoxically, while centromeric function is conserved through eukaryotes, the profile of satellite DNAs in this region is almost always species-specific. We argue that tandem repeats may be advantageous forms of DNA sequences in (peri)centromeres due to concerted evolution, which maintains high intra-array and intrapopulation sequence homogeneity of satellite arrays, while allowing rapid changes in nucleotide sequence and/or composition of satellite repeats. This feature may be crucial for long-term stability of DNA-protein interactions in centromeric regions.
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              Wolbachia endobacteria depletion by doxycycline as antifilarial therapy has macrofilaricidal activity in onchocerciasis: a randomized placebo-controlled study

              In a randomized, placebo-controlled trial in Ghana, 67 onchocerciasis patients received 200-mg/day doxycycline for 4–6 weeks, followed by ivermectin (IVM) after 6 months. After 6–27 months, efficacy was evaluated by onchocercoma histology, PCR and microfilariae determination. Administration of doxycycline resulted in endobacteria depletion and female worm sterilization. The 6-week treatment was macrofilaricidal, with >60% of the female worms found dead, despite the presence of new, Wolbachia-containing worms acquired after the administration of doxycycline. Doxycycline may be developed as second-line drug for onchocerciasis, to be administered in areas without transmission, in foci with IVM resistance and in areas with Loa co-infections.
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                Author and article information

                Contributors
                carlow@neb.com
                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group UK (London )
                2045-2322
                16 July 2019
                16 July 2019
                2019
                : 9
                Affiliations
                [1 ]ISNI 0000 0004 0376 1796, GRID grid.273406.4, New England Biolabs, ; Massachusetts, USA
                [2 ]ISNI 0000 0004 1937 0722, GRID grid.11899.38, Department of Parasitology, Institute of Biomedical Sciences, , University of São Paulo, ; São Paulo, Brazil
                [3 ]ISNI 0000 0001 2288 3199, GRID grid.29273.3d, Department of Microbiology and Parasitology, , University of Buea, ; Buea, Cameroon
                Article
                46550
                10.1038/s41598-019-46550-9
                6635353
                31311985
                © The Author(s) 2019

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

                Funding
                Funded by: FundRef https://doi.org/10.13039/100004774, New England Biolabs (NEB);
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                © The Author(s) 2019

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                diagnostic markers, data mining

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