Monitoring Infected Reticulocytes During Murine Malaria Infection

Abstract

Abstract Malaria infection is a major cause of mortality worldwide. According to the 2022 World Health Organization Malaria Report, malaria caused approximately 247 million infections and 619,000 deaths in 2021, with more than 470,000 children dying of malaria. The gold-standard for measuring parasitemia is counting infected red blood cells (RBCs) by eye using Giemsa staining whole blood smears. This method is time consuming and is subject to error by both who is counting, the number of cells counted, and which frames are selected for counting. Automating the process of measuring malaria infection by flow cytometry decreases both the time required to obtain accurate parasitemia counts and the possible error on behalf of the researcher. Hundreds of thousands of cells can be counted by flow cytometry in a shorter period of time. Animal models of malaria infection are widespread and well-characterized. The rodent malaria strain P. berghei has shown preferential infection of reticulocytes over mature RBCs. Here we outline a method of measuring P. berghei blood stage infection in reticulocytes and mature RBCs and comparing the results to Giemsa stained whole blood smears. Using commercially available P. berghei ANKA expressing green-fluorescent protein (GFP), we infected wild type mice with 1x106 parasitized RBCs by intraperitoneal injection. Whole blood samples were collected daily starting day 3 post infection through day 7 by tail vein collection. Random and representative fields of Giemsa stained peripheral smears were selected to measure the percentage of parasitemia in mature RBCs and reticulocytes for a total cell count of approximately 1000. For flow cytometry, whole blood was incubated in 500uL of 1X SYBR Green I Nucleic Acid Gel Stain in PBS at 37°C for 20 minutes in the dark, washed with PBS, and stained with anti-CD71 to label reticulocytes. During the early stages of infection, the reticulocyte percentage of infected animals is not significant from uninfected animals, approximately 1.86%. As parasitemia increases over time, although the percentage of total reticulocytes decreases, the percentage of infected reticulocytes increases. On day 3 through 7 for one experimental replicate, day 3 demonstrated 1.86% total reticulocytes, and of these 2.56% reticulocytes were infected; day 4 had 0.66% total reticulocytes, and of these 32.92% were infected; day 5 showed 0.18% reticulocytes with 71.81% infected reticulocytes; on day 6 there were 0.04% reticulocytes with 75.44% infected reticulocytes; and day 7 had 0.01% reticulocytes with 69.56% infected reticulocytes. There was a strong correlation (R2 = 0.9147) when comparing flow cytometry to measure parasitemia with counting parasitemia by peripheral smear. This flow cytometry panel is an excellent tool to assess malarial infection of reticulocytes compared to mature RBCs and can be applied to other malarial species while decreasing bias, error and time needed to conduct the experiment.