Maintaining Plasmodium falciparum gametocyte infectivity during blood collection and transport for mosquito feeding assays in the field

Anopheles stephensi Sind-Kasur Nijmegen strain [18] (Radboudumc) were reared at 30 °C, and Anopheles coluzzii (Burkina Faso and The Gambia) were reared at 27 °C. All were kept at ~ 70–80% relative humidity (RH) on a 12-h day/night cycle and were fed on 5–10% glucose. Female mosquitoes between 2 and 6 days post emergence were used for all mosquito feeding assays. After feeding on gametocyte infected blood, mosquitoes were stored either at 26 °C (Radboudumc) or 27 °C (Burkina Faso and The Gambia).

SMFAs were performed at Radboudumc, Nijmegen, The Netherlands, using cultured Plasmodium falciparum gametocytes as previously described [19]. In brief, P. falciparum was cultured at 37 °C in an automated incubator under a continuous gas flow of 4% CO₂, 3% O₂ and 93% N₂. Gametocytes were produced from asynchronous cultures (day 0, 1% parasitaemia and 5% red blood cells), which were harvested at day 15. Gametocyte infected blood meals were prepared by adding 900 µl mature stage 5 gametocyte culture to 600 µl packed red blood cells, this was briefly centrifuged, the supernatant removed and 600 µl human serum added. For the long-term storage experiments, gametocyte infected blood meals were either fed immediately to mosquitoes or were stored for 4 h in a tube in a thermos flask filled with water at a range temperatures, measured using a calibrated probe thermometer accurate to ± 0.2 °C (Traceable® Ultra digital thermometer, VWR 620-2079) before feeding to mosquitoes. The water temperatures chosen were either the temperature hypothesized to be the optimal gametocyte storage temperature (the temperature of the human body), 37 °C (range: 36.9–37.2 °C) or an arbitrarily selected lower (35.5 °C [range: 35.4–35.6 °C]) or higher (37.8 °C [range: 37.8–37.8 °C]) temperature. Thermos flasks (Stainless king food flask 470 ml, Thermos) were stored either at room temperature (RT; mean 21.3 °C, range: 20.4–22.1 °C) or in an incubator set at 32 °C (range: 31.9–32.2 °C) or 42 °C (range: 41.4–42.1 °C). These storage temperatures were selected to reflect the range of temperatures that might be experienced during thermos flask storage in a malaria endemic county. The exact thermos storage temperature and water temperatures are presented in Additional file 1: Table S1. For the short-term storage experiments, gametocyte infected blood meals were stored for 15 min at RT or in a heat block at a range of temperatures (30 °C, 35.5 °C, 38 °C, 40 °C or 42 °C) before feeding to mosquitoes. For all SMFAs, female mosquitoes were offered the infected blood meal in glass mini-feeders (300 μL) attached to a circulating water bath set at 39 °C and allowed to feed in the dark for 15 min [19]. A total of 30–60 mosquitoes in 2 or 3 cups were used for each experimental condition. Mosquitoes were maintained on 5% glucose and dissected 6–8 days after feeding and the number of developed oocysts per mosquito was determined by microscopy after 1% mercurochrome midgut staining.

The Burkina Faso study was approved by the London School of Hygiene and Tropical Medicine ethics committee (Review number: 14724), the Centre National de Recherche et de Formation sur le Paludisme institutional review board (Deliberation N° 2018/000,002/MS/SG/CNRFP/CIB) and the Ethics Committee for Health Research in Burkina Faso (Deliberation N° 2018–01-010). The Gambia study was approved by the London School of Hygiene and Tropical Medicine ethics committee (Review number:15993) and by The Gambia Government/MRC Joint Ethics Committee (Review number:1621). All participants gave informed consent before inclusion in the studies.

Gametocyte positive human blood samples were obtained from individuals as part of ongoing studies in Burkina Faso and The Gambia. In Burkina Faso, participants were microscopy-positive gametocyte carriers aged 10–15 years old, recruited from screening campaigns in the Saponé health and demographic surveillance system area, ~ 45 kms Southwest of Ouagadougou. In The Gambia, participants were microscopy-positive gametocyte carriers aged > 2 years, passively recruited from four health facilities (Basse Hospital, Sabi, Sotuma Sere, Gambissara) in the South Bank of the Upper River Region in The Gambia.

Statistical analysis was performed using GraphPad Prism (ver. 8). Mosquito infectivity in SMFAs was analysed by comparing groups with the Kruskal–Wallis test comparing each experimental condition to the control with Dunns multiple comparison test. Mosquito infectivity in DMFAs was analysed by comparing oocyst intensity (average oocysts per mosquito) or prevalence of infection (% of infected mosquitoes) by Wilcoxon matched-pairs signed rank test. Agreement between the immediate DMFAs or DMFAs with stored gametocytes was analysed using Spearmans correlation of % mosquitoes infected before and after gametocyte storage and Bland–Altman analysis to visualize and test whether systematic bias in infection rates occurred between the two methods.

To determine the optimal storage conditions for cultured stage 5 gametocytes that maintains their infectivity, SMFAs were performed where two conditions were evaluated; (i) the starting water temperature in the thermos flask and (ii) the temperature the sealed thermos flask was stored at. In a series of experiments, mosquito blood meals were prepared with mature stage 5 gametocyte cultures and either fed immediately to mosquitoes (control) or stored in a thermos flask for 4 h. The thermos flasks were filled with water at either 37.8 °C, 37 °C or 35.5 °C and after addition of the mosquito blood meal the thermos flasks were sealed and stored at either room temperature (RT), 32 °C or 42 °C. The stability of the water temperature was evaluated 2 and 4 h after initial filling of the thermos for all experiments (Fig. 1 and Additional file 1: Table S1). The impact of storing the cultured gametocytes in the thermos flask was assessed by comparing the level of transmission, as measured by the intensity of mosquito oocyst infection, in the immediate control SMFA to the SMFA performed with the stored gametocytes.

During the 4 h of storage, the thermos flasks were opened once to measure the temperature before being sealed again and the temperature measured a final time after 4 h. The water temperatures remained relatively stable during storage, with a greater reduction in water temperature observed when the thermos was stored at RT (mean change  −3.08 °C) compared to 32 °C (mean change  −1.59 °C) or 42 °C (mean change 0.04 °C) (Fig. 1 and Additional file 1: Table S1). There was no reduction in transmission when the gametocyte infected blood meal was stored for up to 4 h in a thermos containing water at 35.5 °C and the thermos flask was stored between RT (~ 21.3 °C) and 32 °C. However, when the thermos was stored at a higher temperature (42 °C) there was a statistically significant reduction in intensity of oocyst infection in 50% of the experiments (Fig. 2 and Table 1). When the water in the thermos was higher, at 37 °C or 37.8 °C, a statistically significant reduction in transmission was occasionally observed after gametocyte infected blood meal storage, even when the thermos was kept between RT (~ 21.3 °C) and 32 °C. This was more frequent when the water was 37.8 °C (Table 1 and Fig. 2). The percentage of mosquitoes infected in each experiment followed the same trend as intensity of infection, but was less affected by the storage conditions. These results indicate that in order to maintain infectivity of gametocytes they should be stored in a thermos filled with water that is 35.5 °C and the thermos should be stored between 21.3 °C and 32 °C. In 3 experiments there was an increase in oocyst intensity. This was only observed in 1/6 experiments using the above proposed optimal conditions.

Since the optimal temperature for 4 h of gametocyte storage was determined to be 35.5 °C, lower than expected, the standard temperature used in the membrane feeders for SMFAs (37 °C) was examined to determine whether this might also have a negative effect on gametocyte transmission to mosquitoes. To evaluate this in a series of experiments, cultured stage 5 gametocyte blood meals were stored for 15 min (the standard duration of mosquito feeding in a SMFA) at either 35.5 °C (safe temperature for 4-h storage) or a range of higher and lower temperatures (RT, 30 °C, 38 °C, 40 °C and 42 °C).

Short-term storage of the gametocyte blood meal for 15 min at 42 °C resulted in significant reduction in transmission, but short-term storage at temperatures lower than this (40 °C, 38 °C, 30 °C and RT) did not have a negative effect (Fig. 3). This indicates that the temperature of the membrane feeders should not exceed 40 °C for a 15-min mosquito feed, but lower temperatures (as low as 21.3 °C) are not detrimental for this short duration.

To assess if the optimal thermos storage conditions determined using cultured gametocytes were also suitable for storage of natural gametocytes in field conditions, paired experiments (n = 47 in Burkina Faso and n = 16 in The Gambia) were performed where blood was collected from gametocyte infected individuals and either fed immediately to mosquitoes in a DMFA or stored in the thermos flask for up to 4 h before DMFA. The starting temperature of the water in the thermos flask was 35.5–36 °C, and the thermos flasks were stored at ambient lab temperature or in an air-conditioned car (range: 25.5–29 °C). The prevalence of infected mosquitoes and the intensity of mosquito infection was similar for the DMFAs performed immediately compared to the DMFAs performed after 4 h storage in a thermos flask (Fig. 4a, b). When comparing results from the two assays, there was a strong correlation (Spearman’s r = 0.9431, p < 0.0001) and good agreement using the Bland–Altman analysis (mean difference in mosquito infection rate = − 0.07%) (Fig. 4c, d).