Proceedings of an expert workshop on community agreement for gene drive research in Africa - Co-organised by KEMRI, PAMCA and Target Malaria.

Gene drive research is progressing towards future field evaluation of modified mosquitoes for malaria control in sub-Saharan Africa. While many literature sources and guidance point to the inadequacy of individual informed consent for any genetically modified mosquito release, including gene drive ones, (outside of epidemiological studies that might require blood samples) and at the need for a community-level decision, researchers often find themselves with no specific guidance on how that decision should be made, expressed and by whom. Target Malaria, the Kenya Medical Research Institute and the Pan African Mosquito Control Association co-organised a workshop with researchers and practitioners on this topic to question the model proposed by Target Malaria in its research so far that involved the release of genetically modified sterile male mosquitoes and how this could be adapted to future studies involving gene drive mosquito releases for them to offer reflections about potential best practices. This paper shares the outcomes of that workshop and highlights the remaining topics for discussion before a comprehensive model can be designed

Proceedings of an expert workshop on community agreement for gene drive research in Africa - Co-organised by KEMRI, PAMCA and Target Malaria.

Gene drive research is progressing towards future field evaluation of modified mosquitoes for malaria control in sub-Saharan Africa. While many literature sources and guidance point to the inadequacy of individual informed consent for any genetically modified mosquito release, including gene drive ones, (outside of epidemiological studies that might require blood samples) and at the need for a community-level decision, researchers often find themselves with no specific guidance on how that decision should be made, expressed and by whom. Target Malaria, the Kenya Medical Research Institute and the Pan African Mosquito Control Association co-organised a workshop with researchers and practitioners on this topic to question the model proposed by Target Malaria in its research so far that involved the release of genetically modified sterile male mosquitoes and how this could be adapted to future studies involving gene drive mosquito releases for them to offer reflections about potential best practices. This paper shares the outcomes of that workshop and highlights the remaining topics for discussion before a comprehensive model can be designed

Natural Leishmania donovani/Leishmania aethiopica hybrids identified from Ethiopia

Natural hybridization events have been demonstrated between closely and distantly related Leishmania groups despite a predominantly clonal and endogamically sexual mode of reproduction. Here we report the first natural hybrid between Leishmania aethiopica and Leishmania donovani, as evidenced from the analysis of several clones from strain MHOM/ET/94/ABAUY. Targeted species-identification PCRs revealed the presence of both genotypes, and amplified fragment length polymorphisms indicated that the clones are genetically in an intermediate position between both parental species, being more closely related to L. aethiopica. The possible scenario facilitating hybrid formation is not clear, but is discussed in relation to epidemiological data

Leishmania AFLP: paving the way towards improved molecular assays and markers of diversity

Diversity, phylogenetic, and population genetic studies of the genus Leishmania, causative agent of leishmaniasis, nowadays generally involves multilocus microsatellite and multilocus sequence typing. Even though these are well established and useful applications, amplified fragment length polymorphisms (AFLP) can provide complementary information. In addition, as the technique essentially probes the entire genome at random, without prior sequence knowledge, it is ideally suited as a screening tool for molecular markers linked with biological and clinical traits. We developed an AFLP protocol adapted to the Leishmania genome, tested its repeatability, and validated it on a panel of samples from the Leishmania donovani complex previously analyzed by multiple molecular tests. The technique proved highly reproducible, and showed that genetic relationships between L. donovani strains generally reflect geographic distance. Four main groups were identified: L. infantum, African L. donovani, Indian L. donovani, and a mixed group consisting of L. donovani from India and Africa. Results were highly congruent with previous analyses on essentially the same sample set, indicating that the developed assay produces trustworthy data. This opens possibilities for application in studies of speciation and population dynamics. Moreover, it allows random screening of the entire Leishmania genome for linkage with biological and clinical parasite properties, such as fitness, drug resistance, and disease profile

Recombinant expression of trypanosome surface glycoproteins in Pichia pastoris for the diagnosis of Trypanosoma evansi infection

Serodiagnosis of surra, which causes vast economic losses in livestock, is still based on native antigens purified from bloodstream form Trypanosoma (T.) evansi grown in rodents. To avoid the use of laboratory rodents in antigen preparation we expressed fragments of the invariant surface glycoprotein (ISG) 75, cloned from T. brucei gambiense cDNA, and the variant surface glycoprotein (VSG) RoTat 1.2, cloned from T. evansi gDNA, recombinantly in Pichia (P.) pastoris. The M5 strain of this yeast has an engineered N-glycosylation pathway resulting in homogenous Man5GlcNAc2 N-glycosylation which resembles the predominant Man9-5GlcNAc2 oligomannose structures in T. brucei. The secreted recombinant antigens were affinity purified with yields of up to 10mg and 20mg per liter cell culture of rISG 7529-465-E and rRoTat 1.223-385-H respectively. In ELISA, both recombinant proteins discriminated between pre-immune and immune serum samples of 25 goats experimentally infected with T. evansi. The diagnostic potential of rRoTat 1.223-385-H but not of rISG 7529-465-E was confirmed with sera of naturally infected and control dromedary camels. The results suggest that rRoTat 1.223-385-H expressed in P. pastoris requires further evaluation before it could replace native RoTat 1.2 VSG for serodiagnosis of surra, thus eliminating the use of laboratory animals for antigen production

Universal PCR assays for the differential detection of all Old World Leishmania species

For the epidemiological monitoring and clinical case management of leishmaniasis, determination of the causative Leishmania species gains importance. Current assays for the Old World often suffer from drawbacks in terms of validation on a geographically representative sample set and the ability to recognize all species complexes. We want to contribute to standardized species typing for Old World leishmaniasis. We determined the ribosomal DNA internal transcribed spacer 1 sequence of 24 strains or isolates, and validated four species-specific polymerase chain reactions (PCRs) amplifying this target. They discriminate L. aethiopica, L. tropica, L. major, and the L. donovani complex, use the same cycling conditions, and include an internal amplification control. Our PCRs amplify 0.1 pg of Leishmania DNA, while being 100% specific for species identification on an extensive panel of geographically representative strains and isolates. Similar results were obtained in an endemic reference laboratory in Kenya. Species could also be identified in clinical specimens. The presented PCRs require only agarose gel detection, and have several other advantages over many existing assays. We outline potential problems, suggest concrete solutions for transferring the technique to other settings, and deliver the proof-of-principle for analyzing clinical samples