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5. Molecular determination of Plasmodium falciparum genetic identity in infected Anopheles mosquitoes

Author

Bassiouni, Sarah, Chiumia, Martin, Vareta, Jimmy, Mzilahowa, Themba, Taylor, Terrie, and Seydel, Karl

Subjects
parasitic diseases
Abstract

As malaria transmission wanes, an increasing number of individuals harbor asymptomatic infections that are not typically detected by conventional rapid diagnostic methods. This presents distinct challenges as interventions shift from malaria control to elimination in endemic settings. To identify these asymptomatic carriers and to better understand transmission patterns on the individual human level, it is important to identify individual Plasmodium parasites in both humans and mosquitoes. Although previous work has identified individual parasites in human blood samples, this has not yet been replicated in mosquitoes. In this proof of concept experiment, we focused on genetic identification of individual Plasmodium ring and trophozoite stage parasites in blood-fed Anopheles mosquito midguts. We established in vitro levels of detection of individual Plasmodium parasites in human blood samples using four different parasitemias (10000, 1000, 100, and 10 parasites/µL) and four mixed ratios of (80:20, 20:80, 90:10, and 10:90) of both the standard laboratory Plasmodium strain 3D7 and a clinical isolate at the ring and trophozoite stages using qPCR. We then conducted Anopheles mosquito membrane feeds using the same four levels of Plasmodium parasitemias and four mixed ratios of the genetically distinct parasites to determine a pragmatic level of monoclonal and multiclonal detection specificity within the mosquito midgut. Fed mosquitoes were dissected and DNA extracted from the abdomens. qPCR for Plasmodium falciparum L-lactate dehydrogenase (PfLDH) DNA was used to determine presence of Plasmodium. Samples positive for PfLDH were then characterized at six previously identified single nucleotide polymorphisms. Data regarding sensitivity will be presented; preliminary results indicate that although in vitro detection is quite sensitive (to the 100 parasites/µL level), in vivo detection at low parasitemia (

Published
2022
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7. Human cerebral malaria and Plasmodium falciparum genotypes in Malawi

Author

Milner Danny A, Vareta Jimmy, Valim Clarissa, Montgomery Jacqui, Daniels Rachel F, Volkman Sarah K, Neafsey Daniel E, Park Daniel J, Schaffner Stephen F, Mahesh Nira C, Barnes Kayla G, Rosen David M, Lukens Amanda K, Van-Tyne Daria, Wiegand Roger C, Sabeti Pardis C, Seydel Karl B, Glover Simon J, Kamiza Steve, Molyneux Malcolm E, Taylor Terrie E, and Wirth Dyann F

Subjects
Plasmodium falciparum, Cerebral malaria, Genotyping, Molecular barcode, Histopathology, Autopsy, Arctic medicine. Tropical medicine, RC955-962, Infectious and parasitic diseases, RC109-216
Abstract

Abstract Background Cerebral malaria, a severe form of Plasmodium falciparum infection, is an important cause of mortality in sub-Saharan African children. A Taqman 24 Single Nucleotide Polymorphisms (SNP) molecular barcode assay was developed for use in laboratory parasites which estimates genotype number and identifies the predominant genotype. Methods The 24 SNP assay was used to determine predominant genotypes in blood and tissues from autopsy and clinical patients with cerebral malaria. Results Single genotypes were shared between the peripheral blood, the brain, and other tissues of cerebral malaria patients, while malaria-infected patients who died of non-malarial causes had mixed genetic signatures in tissues examined. Children with retinopathy-positive cerebral malaria had significantly less complex infections than those without retinopathy (OR = 3.7, 95% CI [1.51-9.10]).The complexity of infections significantly decreased over the malaria season in retinopathy-positive patients compared to retinopathy-negative patients. Conclusions Cerebral malaria patients harbour a single or small set of predominant parasites; patients with incidental parasitaemia sustain infections involving diverse genotypes. Limited diversity in the peripheral blood of cerebral malaria patients and correlation with tissues supports peripheral blood samples as appropriate for genome-wide association studies of parasite determinants of pathogenicity.

Published
2012
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13. GENOMIC TOOLS REVEAL TRENDS IN PLASMODIUM FALCIPARUM PARASITE DIVERSITY ACROSS TRANSMISSION GRADIENTS WORLDWIDE

Author

Daniels, Rachel Fath, Deme, Awa B., Bei, Amy K., Bennett, Adam, Silumbe, Kafula, Chalwe, Victor, Kamuliwo, Mukalwa, Hamainza, Busiku, Yukich, Josh, Chizema, Elizabeth, Earle, Duncan, Huijben, Silvie, Aide, Pedro, Galatas, Beatriz, Lukens, Amanda K., Dondorp, Arjen M., Chang, Hsiao-Han, Eziefula, Alice, Tadesse, Fitsum G., Grignard, Lynn, Baro, Nicholas K., Morton, Lindsay, Charles, Macarthur, Vareta, Jimmy A., Ahmad, Rushdy, Taylor, Terrie E., Buckee, Caroline, Teun Bousema, Drakeley, Chris, Golightly, Linnie, Eisele, Thomas P., Steketee, Richard W., Miller, John M., Fairhurst, Rick M., Aponte, John, Alonso, Pedro L., Mayor, Alfredo, Milner, Danny A., Udhayakumar, Venkatachalam, Ndiaye, Daouda, Wirth, Dyann F., Hartl, Daniel L., and Volkman, Sarah K.

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