TRA estimates based on oocyst counts and luminescence measures were highly related (Figure 2A). Deviations from this linear trend were more apparent when both methods estimated TRA at <60%, as reflected by the size of the CIs.
A stochastic element in measuring TRA is well appreciated [5, 6, 22] and a cut-off for reproducible TRA results of >80% is commonly used [6, 23]. Where TRA ≥80% was measured by either method the mean difference between luminescence and microscopy based TRA estimates was 4.0%, and CIs were equally precise for both methods. Across the range of TRAs, CIs were generally wider for luminescence (mean interval, 20.4 percentage points) than for microscopy (mean interval, 15.0 percentage points). TRA estimates based on the luminescence assay for pooled mosquitoes were similarly representative of microscopy-based estimates (Figure 2B). When test agents caused a TRA ≥80%, the mean difference between microscopy- and pool-based TRA estimates was 3.6% and 3.0% for pools of 5 and 10, respectively. Supplementary figure 2A shows the results of all pool-based TRA estimates relative to microscopy values if only single pools are used in their calculation (instead of all 3).
In our large set of blinded experiments, we determined the TRA of different blocking and non-blocking concentrations of 85RF45.1 mAb and serum IgG. TRA was calculated using microscopy and luminescence of individual mosquitoes and pools of mosquitoes. We observed excellent agreement between TRA estimates (figures 2C and 2D), with only one of the weakest dilutions of serum IgG luminescence giving non-significantly higher TRA estimates derived from pools of 5 mosquitoes.
Luminescent P. falciparum in the SMFA
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Figure 2. Microscopy- and luminescence-based assessments of the transmission-reducing activity (TRA) of transmission effective mAb (Pfs45.1), and serum immunoglobulin G (IgG).
All TRA calculations were made using human serum controls (Supplementary Figure 1) A. TRA calculated separately, using mean oocyst intensity and mean luminescence intensity from individual mosquitoes from all feeds in which a transmission-reducing agent was used (n=46). R2 was calculated based on deviation from a perfect linear association (i.e. x=y). The mean widths of the 95% CIs in which either estimate was ≥80% was 3.86 percentage points and 3.61 percentage points for luminescence and microscopy, respectively. B. TRA calculated separately using mean oocyst intensity and mean luminescence intensity from pools of 5 (print = light grey, online = blue) and 10 (print = dark grey, online = red) mosquitoes from all feeds for which a transmission-reducing agent was used and from which the minimum number of pools were analysed (pools of 5, n=33; pools of 10, n=38). R2 was calculated based on deviation from a perfect linear association (i.e. x=y). For data presentation, 1% TRA
Discussion
Our primary finding is that luciferase-expressing P. falciparum strains can be used effectively in the SMFA to provide an objective, scalable, and accurate measure of infection intensity in groups of individual and pooled mosquitoes. The luminescence assay discriminated accurately between infected and uninfected mosquitoes. The mean luminescence intensity of individual and pooled undissected mosquitoes correlated strongly with mean oocyst intensity in the same feeds, and the strength of this relationship was reflected in the close association of TRA estimates made by both methods.
Precise estimates of TRA depend on experiment replication and an adequate number of mosquito observations [5, 6]. These requirements are often not met in the SMFA as it is routinely conducted; improvements in throughput and readout are needed to allow more-robust and more-scalable assessments of TRA. We present the first approach that eliminates the requirement for mosquito dissections and allows groups of individual or pooled mosquitoes to be examined for the average intensity of infection. Previous attempts to increase SMFA efficiency continued to rely on manual mosquito dissection and semiautomated oocyst visualisation, with modest impact on SMFA throughput [7, 8]. Accepting the loss of information at the individual mosquito level, we show that the averaged luminescence of groups or pools of mosquitoes provides an accurate proxy for mean infection intensity. The most scalable approach presented in the current study was the screening of pools of mosquitoes. Although luminescence intensity was generally slightly lower for pools than for individual mosquitoes, possibly because of the slightly longer homogenisation required during pool processing, luminescence intensity was similarly predictive of oocyst intensity for all methods. When examining 3 pools from each feed and assaying each pool in triplicate, 10 feeds can be evaluated simultaneously on a single assay plate. We observed concordant results if single pools of mosquitoes were examined, indicating that mosquito homogenisation can be made more scalable if fewer pools are examined per feed. Further improvements in throughput and in the standardisation of mosquito processing might be achieved if pools are homogenised using plate-based bead beating (Supplementary Materials); this technique might also be used to scale-up individual mosquito processing if details of infection prevalence are desirable. Other clear benefits of luminescence-based outcomes are that they are recorded objectively and stored automatically. With the combined advantages of the luminescence assay we estimate a five to ten fold increase in our SMFA throughput capacity (Supplementary Materials).
The luciferase assay can detect increases in oocyst size and maturation, as indicated by the higher luminescence intensity on days 8 and 9 after infection, compared with that on day 7. Inevitable variations in the productivity of concomitant oocysts limit our ability to accurately interpret oocyst number from an indirect measure of luminescence intensity for individual mosquitoes, which is likely only possible using visualisation techniques [7].
Luminescent P. falciparum in the SMFA
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However, we find no evidence that these variations affect luciferase-based TRA estimates, which consistently mirror those based on microscopical read-outs. Because size differences are more likely in high-intensity infections, where nutrient and space competition may be more pronounced, very high control oocyst intensities may need to be avoided to ensure an accurate correlation between mean luminescence and oocyst intensity. It could be argued, though, that because luciferase production reflects sporozoite proliferation more directly, luminescence intensity may provide a less abstract measure of TRA than oocyst intensity.
The disadvantages of the traditional SMFA in this respect have been discussed previously [5, 8]. Luminescence-based outcomes are particularly suitable for studies where changes in oocyst size or maturation and not solely their presence are of interest [8] and for assessments of other aspects of sporogonic development, such as haemolymph or salivary gland infection, where parasite loss during standard investigations is a concern.
In the conventional SMFA with microscopy-based readouts the threshold for transmission blockage is commonly set at 80% transmission reduction [23], not because vaccines or drugs with lower levels of TRA may not be of value [24] but because there is considerable variation in estimates of low level TRA, and only high level transmission reduction can be reproducibly measured [6, 22]. As anticipated, in our experiments, CIs were narrowest and agreement between luminescence- and microscopy-based estimates were greatest in the transmission blocking range. Estimating lower levels of the TRA requires a larger number of mosquito observations, ideally collected through a study in which the activity of compounds or test sera is assessed against a range of control oocyst densities[5]. Because of its scalability and strong association with oocyst prevalence and intensity, the proposed luminescence assay can facilitate these repeated assessments of large mosquito numbers that may be operationally unattractive when relying on microscopy. Despite the value of our approach, there are experiments where microscopy remain of indisputable value. These include experiments with A. gambiae mosquitoes, where the use of the NF54HT GFP-Luc parasite line is precluded, and investigations where oocyst enumeration is of specific interest.
A high-throughput, semiautomated SMFA could significantly increase the efficiency of screening drugs and vaccines with potential effects on Plasmodium transmission, which have taken a central role in plans to achieve malaria eradication. The results of the current study represent a scalable alternative to traditional SMFA techniques that may also increase the operational feasibility of population-level trials with transmission-blocking vaccines, by complementing direct-feeding assessments with scalable assessments of transmission blocking immunity [25].
Foundation (AFIRM, grant number OPP1034789) and PATH Malaria Vaccine Initiative (MVI). TB is further supported by European FP7 project REDMAL (#242079) and a Marie Curie Career Integration Grant from the European Community’s Seventh Framework Programme (SIGNAL, PCIG12-GA-2012-333936). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Luminescent P. falciparum in the SMFA