The majority of human clinical blood samples used in this study were originally submitted to the CDC Parasitic Diseases Branch for confirmatory diagnosis of parasitic infections. Following diagnosis, samples were de-identified and frozen in 200 μL aliquots at − 80 °C for later use. De-identification involved the collector of the specimens providing an aliquot to the authors of this study in an ambiguously marked container (e.g., “P. falciparum specimen 1”) so that its linkage back to the patients was not possible. Samples acquired in this manner include the following: P. falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, Babesia microti, Babesia divergens-like variant MO1, T. cruzi (HIV-positive and HIV-negative clinical samples), Loa loa, and NPF (no parasite found)—used as negative controls. A blood spot on filter paper collected in the Democratic Republic of Congo from a person with Mansonella perstans microfilaremia was provided by Dr. Vita Cama of the CDC Parasitic Diseases Branch. Acute T. cruzi infection EDTA blood DNA extracts were generously provided by Dr. Stella Chenet and Dr. Maria Isabel Jercic of the Institute of Public Health, Chile (Santiago). B. divergens was obtained from the infected blood of laboratory-raised gerbils, and Babesia duncani was obtained from the infected blood of laboratory-raised guinea pigs, routinely maintained in CDC’s animal facility (Table (Table1).1). Bioinformatic analysis confirmed that the restriction enzyme cut sites were present in all the respective animal species for all relevant animal samples utilized here. For some rare blood-borne parasites (Plasmodium knowlesi and Brugia malayi), either animal blood or human blood collected during previous studies and stored at − 80 °C were used. Rare blood parasites that could not be acquired as true clinical samples at the time of the study were recreated by spiking uninfected human blood with cultured parasites—Leishmania infantum, Leishmania donovani, T. cruzi, and Trypanosoma brucei subsp. rhodesiense cultures were added to whole human blood at a ratio of 1:100. All blood samples were collected into EDTA anticoagulant. Full details regarding specimen source, matrix, original parasite identification method, and DNA extraction method are provided in Table Table1.1. Simulated mixed parasite infections were prepared according to the descriptions in Table S1, which can be found in Supplementary File S1 on page 4.

Host, source, and original identification method of samples used in this study

w/s with Sanger sequencing of PCR product, CDC Centers for Disease Control and Prevention, FR3 Filariasis Research Reagent Resource Center, PDB Parasitic Diseases Branch, UGA University of Georgia, NIH National Institutes of Health

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