Refined Method for Droplet Microfluidics-Enabled Detection of Plasmodium falciparum Encoded Topoisomerase I in Blood from Malaria Patients

Hede, Marianne Smedegaard; Okorie, Patricia N.; Fruekilde, Signe Kirk; Fjelstrup, Søren; Thomsen, J.; Franch, Oskar; Tesauro, Cinzia; Bugge, Magnus Tobias; Christiansen, Mette; Picot, Stéphane; Lötsch, Felix; Mombo-Ngoma, Ghyslain; Mischlinger, Johannes; Adegnika, Ayôla Akim; Pedersen, Finn Skou; Ho, Yi‐Ping; Petersen, Eskild; Stougaard, Magnus; Ramharter, Michael; Knudsen, Birgitta R.

doi:10.3390/mi6101432

Cited by 8 publications

(4 citation statements)

References 9 publications

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“…A variety of diagnostic tools exists for the detection of malaria in blood (135)(136)(137)(138)(139)(140)(141)(142)(143). Depending on the available resources, it may be feasible to use highly sensitive and specific methods, such as PCR, loop-mediated amplification (LAMP), or enzyme-linked immunosorbent assay (ELISA) (137,141,(143)(144)(145)(146).…”

Section: Diagnosis Of Plasmodium Infections In Non-malaria-endemic Comentioning

confidence: 99%

Imported Malaria in Countries where Malaria Is Not Endemic: a Comparison of Semi-immune and Nonimmune Travelers

et al. 2020

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SUMMARY The continuous increase in long-distance travel and recent large migratory movements have changed the epidemiological characteristics of imported malaria in countries where malaria is not endemic (here termed non-malaria-endemic countries). While malaria was primarily imported to nonendemic countries by returning travelers, the proportion of immigrants from malaria-endemic regions and travelers visiting friends and relatives (VFRs) in malaria-endemic countries has continued to increase. VFRs and immigrants from malaria-endemic countries now make up the majority of malaria patients in many nonendemic countries. Importantly, this group is characterized by various degrees of semi-immunity to malaria, resulting from repeated exposure to infection and a gradual decline of protection as a result of prolonged residence in non-malaria-endemic regions. Most studies indicate an effect of naturally acquired immunity in VFRs, leading to differences in the parasitological features, clinical manifestation, and odds for severe malaria and clinical complications between immune VFRs and nonimmune returning travelers. There are no valid data indicating evidence for differing algorithms for chemoprophylaxis or antimalarial treatment in semi-immune versus nonimmune malaria patients. So far, no robust biomarkers exist that properly reflect anti-parasite or clinical immunity. Until they are found, researchers should rigorously stratify their study results using surrogate markers, such as duration of time spent outside a malaria-endemic country.

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Section: Diagnosis Of Plasmodium Infections In Non-malaria-endemic Comentioning

confidence: 99%

Imported Malaria in Countries where Malaria Is Not Endemic: a Comparison of Semi-immune and Nonimmune Travelers

et al. 2020

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“…Immobilization of DNA forms the foundation of many modern DNA sensor studies including fluorescence microscopic, graphene electronic, and plasmon resonance based methods which may be coupled with a polymerase amplification enabled signal amplification step [ 26 , 27 , 28 ]. A fluorescence microscopy based readout was used to test the effect of DTT on DNA immobilization onto NHS-ester coated glass slides (CodeLink ® Activated slides, Surmodics).…”

Section: Resultsmentioning

confidence: 99%

The Effects of Dithiothreitol on DNA

Fjelstrup

Andersen

Thomsen

et al. 2017

Sensors

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With the novel possibilities for detecting molecules of interest with extreme sensitivity also comes the risk of encountering hitherto negligible sources of error. In life science, such sources of error might be the broad variety of additives such as dithiothreitol (DTT) used to preserve enzyme stability during in vitro reactions. Using two different assays that can sense strand interruptions in double stranded DNA, we here show that DTT is able to introduce nicks in the DNA backbone. DTT was furthermore shown to facilitate the immobilization of fluorescent DNA on an NHS-ester functionalized glass surface. Such reactions may in particular impact the readout from single molecule detection studies and other ultrasensitive assays. This was highlighted by the finding that DTT markedly decreased the signal to noise ratio in a DNA sensor based assay with single molecule resolution.

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“…Using this basic assay setup, we successfully measured TOP1 activity in single human cells [ 24 ] and predicted the CPT cytotoxicity in cancer cell lines [ 26 ]. Additionally, we measured TOP1 activity in biopsies from cancer patients [ 29 ] and proved it possible specifically to detect the presence of human pathogens in crude clinical samples [ 30 , 31 ].…”

Section: Introductionmentioning

confidence: 99%

Simple and Fast DNA Based Sensor System for Screening of Small-Molecule Compounds Targeting Eukaryotic Topoisomerase 1

et al. 2021

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Background: Eukaryotic topoisomerase 1 is a potential target of anti-parasitic and anti-cancer drugs. Parasites require topoisomerase 1 activity for survival and, consequently, compounds that inhibit topoisomerase 1 activity may be of interest. All effective topoisomerase 1 drugs with anti-cancer activity act by inhibiting the ligation reaction of the enzyme. Screening for topoisomerase 1 targeting drugs, therefore, should involve the possibility of dissecting which step of topoisomerase 1 activity is affected. Methods: Here we present a novel DNA-based assay that allows for screening of the effect of small-molecule compounds targeting the binding/cleavage or the ligation steps of topoisomerase 1 catalysis. This novel assay is based on the detection of a rolling circle amplification product generated from a DNA circle resulting from topoisomerase 1 activity. Results: We show that the binding/cleavage and ligation reactions of topoisomerase 1 can be investigated separately in the presented assay termed REEAD (C|L) and demonstrate that the assay can be used to investigate, which of the individual steps of topoisomerase 1 catalysis are affected by small-molecule compounds. The assay is gel-free and the results can be detected by a simple colorimetric readout method using silver-on-gold precipitation rendering large equipment unnecessary. Conclusion: REEAD (C|L) allows for easy and quantitative investigations of topoisomerase 1 targeting compounds and can be performed in non-specialized laboratories.

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Refined Method for Droplet Microfluidics-Enabled Detection of Plasmodium falciparum Encoded Topoisomerase I in Blood from Malaria Patients

Cited by 8 publications

References 9 publications

Imported Malaria in Countries where Malaria Is Not Endemic: a Comparison of Semi-immune and Nonimmune Travelers

Imported Malaria in Countries where Malaria Is Not Endemic: a Comparison of Semi-immune and Nonimmune Travelers

The Effects of Dithiothreitol on DNA

Simple and Fast DNA Based Sensor System for Screening of Small-Molecule Compounds Targeting Eukaryotic Topoisomerase 1

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