The latest world malaria report by WHO names four biological threats to malaria interventions, two of which concern P. falciparum directly1. One is the spread of mutations conferring antimalarial drug resistance, particularly against artemisinins in East Africa, and the other is the spread of mutations in the histidine-rich protein (HRP) 2 and 3 genes, which render standard HRP-based rapid tests falsely negative. Simple, fast and cost-effective molecular techniques applied on a large scale are essential to assess the extent of only these two threats. Traditional surveys based on blood samples from patients or volunteers are time-consuming, costly, and complex: they require ethical clearance, logistical preparation, trained medical personnel, recruitment processes, provision of medical care, cooperation with often overburdened health systems, and they may only capture subgroups of the parasites to be studied. Molecular surveillance of Plasmodium based on mosquitoes could overcome many of these issues. Prerequisites for implementation of xenomonitoring for antimalarial drug resistance are sufficient sensitivity and reproducibility.
In the present study, we demonstrate that molecular detection and genotyping of P. falciparum is possible on whole mosquitoes with low-cost and scalable methods for DNA extraction and amplification. Both infected and blood-fed mosquitoes can be used and pooled, facilitating surveillance. Applying this approach to 50 field-caught Anopheles mosquitoes from DR Congo, we show the feasibility for genotyping drug resistance genes PfK13 and PfMDR1. The presented method provides a starting point to pilot local surveillance, e.g., on drug resistance, leveraging the promising properties of xenomonitoring.
Protocols for molecular typing of P. falciparum from mosquitoes are available3,4, but those often use costly commercial DNA extraction kits and require dissection of the mosquito midgut or salivary glands, which is timely and needs trained entomologists. Reports including limits of detection are scarce and rather use uninfected mosquitoes spiked with Plasmodium DNA instead of actual infected mosquitoes. Moreover, available methods do rarely cater for scale-up, which is a requirement for surveillance. In the present study, we used infected mosquitoes to assess the limit of detection of a combined DNA extraction and PCR assay, accounting for the potential challenges of releasing Plasmodium DNA from the mosquito’s interior. We confirmed that our method does not require dissection of mosquitoes to detect Plasmodium DNA. The DNA extraction method is cost-efficient at an estimated cost of 1€ per sample (70% Proteinase K, 20% Chelex®-Resin-100, 10% ddH2O, pipettips, tubes) and only requires pipetting in a new tube once. A yield of 300 µg after extraction of mosquito tissue is in accordance with other reports5.
PCR assays consistently showed high sensitivity for infected Anopheles mosquitoes mimicking single oocyst-infection (limit of detection at 2000–1000× dilutions). Sensitivity for uninfected mosquitoes spiked with an infected “blood meal”, was sufficient (limit of detection at 250–60× dilutions, corresponding with 0.004–0.02% parasitaemia in the blood meal), but considerably lower than for PCR on mosquitoes mimicking single oocyst-infection. The blood meal sample should theoretically contain more P. falciparum DNA copies compared to a single-oocyst infected mosquito, as 1.5 µL blood with 1% parasite density contains roughly 60,000 parasite genome copies (assuming 4,000,000 RBCs/µL blood) versus up to 15,000 copies from sporozoites per oocyst found in lab infected mosquitoes6. These counterintuitive results may be explained by PCR inhibitory effects of hemoglobin remnants after extraction7. Chelex-based extraction does not result in DNA as pure as after silica-based extraction8,9. Inhibiting agents might still be present in the end product and therefore adding Proteinase K and a heating step to 99 °C are essential to degrade potentially problematic proteins in the sample. Of note, adding BSA to the PCR assays is crucial to further limit the effect of potential PCR inhibitors. Silica-based DNA extraction requires the use of commercial spin-columns, which are costly, and result in lower amounts of DNA in the end product9,10. We have therefore favored the use of our in-house Chelex-based extraction protocol7.
The PCR assay for P. falciparum 18s rRNA had a limit of detection twice as low as that for PfK13. This may be due to the Pf 18s rRNA gene being present at 5 copies/genome11, whereas PfK13 occurs only once. However, the PfMDR1 limit of detection being similar to Pf 18s rRNA suggests that the PfK13 PCR has a sequence-specific sensitivity limitation. Our results indicate that for the sensitive detection of P. falciparum DNA in mosquito pools, the Pf 18s rRNA PCR should not be replaced by directly using PfK13 PCR but could be by PfMDR1 PCR.
Our sequencing results of blood samples with parasites carrying different genotypes indicate that the PfK13 and PfMDR1 PCR in combination with Sanger sequencing can detect a mutant parasite clone in a blood meal in a ratio of 1 mutant to 3 wild type single mutations. Mosquitoes may have more than one blood meal from infected hosts12,13, and hosts may carry multiple P. falciparum strains with distinct genetic structure. For surveillance of molecular markers in P. falciparum, we propose a mosquito pool size that corresponds to the probability of containing a maximum of one infected mosquito. Also, a “diagnostic” Pf 18s rRNA PCR should first be done on an initial set of individual mosquitoes to estimate P. falciparum positivity before pooling.
Due to digestion, human DNA is detectable for only 12 h following a blood meal in Culex, up to 48 h in Aedes, and up to 72 h in Anopheles mosquitoes14,15,16. This suggests that Plasmodium DNA in these mosquito species may also only be detectable within this limited time window. On the other hand, (mosquito pools containing) Anopheles mosquitoes may carry Plasmodium DNA for up to 20 days, after the last oocyst matures17, and most Anopheles mosquitoes die before then18. Thus, P. falciparum genotyping results from mixed mosquito pools can theoretically reflect parasite signatures in human hosts from the time of infection for up to 3 weeks after.
We applied our methods on field-caught Anopheles mosquitoes from the high malaria transmission area of Kibali in DR Congo19. Our PCR results revealed that almost 20% of the Anopheles carried P. falciparum DNA. The mutations we found in PfK13, a gene associated with partial artemisinin resistance which recently emerged in East Africa20, are of unknown relevance. Plasmodium falciparum MDR1 mutations at loci 86 and 184 are linked to sensitivity to several antimalarial drugs21, and our observed pattern matches previous observations from the region22,23. These results merely serve as a demonstration of feasibility. One positive pool failed to amplify PfK13. Potentially, storage conditions of these mosquitoes, i.e., at ambient temperature and on silica gel, affected DNA quality, compared to the infected mosquitoes we used for PCR validation that were refrigerated and stored in ethanol. However, drying of mosquitoes is considered to not lead to loss of sensitivity24,25. The unexpected negative PfK13 PCR highlights the need to validate the assays extensively on wild-caught mosquitoes.
A limitation of our study is that the laboratory-infected mosquitoes carried much more oocysts in their midgut (a median of 20) compared to infected field mosquitoes for which reported averages range from 2 to 10 oocysts26,27,28. We diluted the laboratory-infected mosquito DNA with uninfected mosquito DNA to mimic a single-oocyst infection, but this may not have sufficiently represented the low probability of releasing a very low parasite load from mosquito tissue. This holds also true for samples mimicking mosquitoes after an infectious blood meal. Our molecular methods should be validated and further assessed on a model better representing low-infection scenarios and the distribution of all parasite stages. E.g., by setting up and validating Anopheles feeding assays resulting in a low and consistent infection load, by harvesting the mosquitoes at 8, 10, 13, and 15 days post infection, by using P. falciparum field isolates with different drug resistance signatures for the feeding assays, and by assessing other mosquito species after blood feeding with these parasites.
Today’s malaria surveillance is unthinkable without molecular techniques. We present a sensitive and scalable method for molecular surveillance of Plasmodium using anthropophilic mosquitoes, for example to screen for antimalarial resistance markers. Xenomonitoring has a clear potential for up-scaling, and may overcome patient selection bias. With growing molecular biology capacities in Sub-Saharan Africa, opportunities arise for local, fast, cheap and sustainable surveillance using mosquitoes. Implementation should be tailored to local capacities and evaluated for usability and for sensitivity on wild-caught mosquitoes.
