In this study, a method to maintain P. falciparum gametocyte infectivity during blood collection in the field and transportation to the insectary for mosquito feeding assays is presented. The optimal storage conditions preventing gametocyte activation or inactivation were first assessed with cultured gametocytes and then confirmed using natural gametocyte carriers from two malaria endemic settings. The findings corroborate that temperature can play a key role in maintaining the infectivity of P. falciparum gametocytes. Infectivity was not affected when gametocytes were stored in thermos flasks in water at 35.5ºC for up to 4 hours and the thermos flask was stored at ambient temperatures ranging from 21.3ºC to 32ºC.
DMFAs are more commonly used than direct skin feeding assays to assess the infectiousness of gametocyte carriers to mosquitoes, largely because they minimise the discomfort experienced by volunteers, which is particularly important when sampling young children. DMFAs may therefore be more readily acceptable by both local communities and ethics committees (21), especially when repeated assessments of infectivity are made. The relationship between gametocyte density and infection success in mosquitoes has been well studied, but differs slightly by settings (11, 21, 22). These differences could plausibly be due to the different populations, different levels of malaria exposure and resulting levels of transmission-blocking immunity, different mosquito species, or parasite genotypes (8, 9, 11). However, for reliable assessments of infectivity by DMFA it is crucial to minimize technical differences in how the assays are performed between settings (21, 22). Here, evidence is presented that variation in blood storage temperature and duration of storage, as well as feeder temperature, could have a significant impact on transmission.
The results show that the optimal temperature for longer-term storage (4 hours) is 35.5 ºC. The maintenance of gametocyte infectivity at this temperature is consistent with previous studies which showed a drop in temperature of at least 5ºC from the standard 37 °C in the human body, is required to activate P. falciparum gametocytes (12). Also consistent with previous data, our results show that as little as 15 minutes (mimicking the time the gametocytes are present in the feeder) at 40ºC or 42ºC is sufficient to inactivate gametocytes, with transmission being almost completely prevented after storage at 42ºC (13, 14, 16, 23). Taken together, these data suggest that the temperature in the feeders should not reach 40 °C during DMFAs.
With the 4 hour storage experiments, not only temperatures above 40C but also lower temperatures appeared to reduce gametocyte infectivity. It was surprising to find that storing the gametocytes in the thermos flask in water at 37ºC for 4 hours was associated with reduced transmission efficiency in some experiments. This was seen most often when the ambient temperature was high (i.e. 32ºC or 42ºC), and not when it was the typical room temperature in an air-conditioned European laboratory (~ 21.3 °C). The temperature of 37ºC would be hypothesized to be ideal for gametocytes, as it mimics their natural environment in the body, and previous studies have indeed shown that P. falciparum gametocyte activation was prevented when they were maintained at 37ºC for 1 hour (24). In agreement with this, short-term storage for 15 minutes at temperatures up to 38 ºC in our study also did not reduce transmission. This suggests that either the duration of exposure to high temperatures, or the fact that the gametocytes are stored in venous blood collected in lithium heparin anticoagulant, or both, may also be important factors for gametocyte inactivation, although this is not evaluated in this study.
Altogether, the data demonstrates that temperature fluctuations influence gametocyte infectivity, seen most acutely with higher temperatures, including high ambient temperatures, and temperature should thus be controlled when collecting and transporting gametocyte infected blood in regions where temperatures could reach over 40 ºC. An effect of temperature on gametocyte infectivity has been seen before, however, the current study adds value by a large number of replicates in the SMFA with varying thermos and ambient temperatures. This allowed an informed decision on the optimal temperature conditions for use in DMFA experiments using natural gametocytes carriers in the field. With the proposed approach, samples can be transported from more remote settings to the insectary within 4 hours without affecting gametocyte infectivity and thus maintaining assay quality.
This method will facilitate widespread, accurate assessment of malaria transmission dynamics in the field and this understanding will contribute to malaria control strategies as we move towards elimination.